Nucleic acid sample preparation

ABSTRACT

The present invention includes methods, devices and systems for isolating a nucleic acid from a fluid comprising cells. In various aspects, the methods, devices and systems may allow for a rapid procedure that requires a minimal amount of material and/or results in high purity nucleic acid isolated from complex fluids such as blood or environmental samples.

CROSS-REFERENCE

This application claims priority to co-pending U.S. patent applicationSer. No. 14/067,841, filed Oct. 30, 2013, which claims priority to U.S.patent application Ser. No. 13/864,179, filed Apr. 16, 2013, now U.S.Pat. No. 8,603,791, which claims the benefit of U.S. ProvisionalApplication No. 61/624,897, filed Apr. 16, 2012, which application isincorporated herein by reference.

BACKGROUND OF THE INVENTION

Exponentially rapid progress has been made in the field of DNAsequencing in recent years. Methods such as pyrosequencing, ionsemiconductor sequencing and polony sequencing aim to reduce costs to apoint where sequencing a complete genome becomes routine. This isexpected to transform fields as diverse as medicine, renewable energy,biosecurity and agriculture to name a few. However, techniques forisolating DNA suitable for sequencing have not kept pace and there is athreat that this will become a limitation.

SUMMARY OF THE INVENTION

In some instances, the present invention fulfills a need for improvedmethods of nucleic acid isolation from biological samples. Particularattributes of certain aspects provided herein include a total samplepreparation time of less than about one hour, with hands-on time of lessthan about one minute. In some embodiments, the present invention can beused to isolate DNA from dilute and/or complex fluids such as blood orenvironmental samples. In other aspects, the present invention can usesmall amounts of starting material, achieve highly purified nucleicacids, and is amenable to multiplexed and high-throughput operation.

Disclosed herein are methods and devices for quantifying nucleic acid ina sample, comprising: a. applying a sample with a conductivity ofgreater than 100 mS/m to a device, the device comprising an array ofelectrodes capable of establishing an AC electrokinetic field region,the device further comprising at least two chambers, the sample appliedto a first chamber; b. applying known quantities of a nucleic acidstandard to a second chamber; c. establishing a first AC electrokinetichigh field region in the first chamber, the first AC electrokinetic highfield capable of isolating larger nanoparticulate molecular targets; d.establishing a second AC electrokinetic low field in the first chamber,the second AC electrokinetic low field capable of concentrating cells ormicron-sized entities that may be present in the sample; e. establishinga third AC electrokinetic high field to the second chamber, the secondAC electrokinetic high field isolating the molecular nucleic acidstandard applied to the second chamber; f. flushing cells andmicron-sized entities that may be present in the sample from the firstchamber; g. detecting bound nucleic acid signal on the array in thefirst chamber and second chamber; and h. quantifying the bound nucleicacid by comparing the detected signal from the first chamber to detectedsignal from the second chamber.

In some embodiments, the larger nanoparticulate molecular target ischosen from the group consisting of exosomes, high mw nucleic acids,including high mw DNA, oligo-nucleosome complexes, aggregated proteins,vesicle bound DNA, cell membrane fragments and cellular debris. In otherembodiments, the target circulating cell-free biomarker is chosen fromthe group consisting of mutations, deletions, rearrangements ormethylated nucleic acid of circulating DNA, micro RNA, RNA frommicrovesicles or a combination thereof. In still other embodiments, thedetection of the cell-free biomarker provides information useful forcancer diagnosis, cancer prognosis or treatment response in a patient.In yet other embodiments, the tumor cell-free biomarker is associatedwith CNS tumors, neuroblastoma, gliomas, breast cancer, endometrialtumors, cervical tumors, ovarian tumors, hepatocellular carcinoma,pancreatic carcinoma, esophageal tumors, Stoch tumors, colorectaltumors, head and neck tumors, nasopharyngeal carcinoma, thyroid tumors,lymphoma, leukemia, lung cancer, non-small cell lung carcinoma, smallcell lung carcinoma, testicular tumors, kidney tumors, prostatecarcinoma, skin cancer, malignant melanoma, squamous cell carcinoma or acombination thereof. In some embodiments, the tumor cell-free biomarkeris GFAP, VEGF, EGFR, b-FGF, KRAS, YKL-40, MMP-9 or combinations thereof.

In other embodiments, the target biomarker is chosen from the groupconsisting of proteins, lipids, antibodies, high molecular weight DNA,tumor cells, exosomes, nucleosomes and nanosomes. In still otherembodiments, the bound nucleic acid is eluted from the first chamber forfurther characterization. In yet other embodiments, the eluted nucleicacid is amplified or sequenced. In still other embodiments, the sampleis whole blood, serum, plasma, cerebrospinal fluid, body tissue, urineor saliva.

In some embodiments, the AC electrokinetic field is produced using analternating current having a voltage of 1 volt to 40 volts peak-peak,and/or a frequency of 5 Hz to 5,000,000 Hz and duty cycles from 5% to50%. In other embodiments, the conductivity of the sample is greaterthan 500 mS/m. In yet other embodiments, the array of electrodes isspin-coated with a hydrogel having a thickness between about 0.1 micronsand 1 micron. In still other embodiments, the hydrogel comprises two ormore layers of a synthetic polymer. In some embodiments, the hydrogelhas a viscosity between about 0.5 cP to about 5 cP prior tospin-coating. In other embodiments, the hydrogel has a conductivitybetween about 0.1 S/m to about 1.0 S/m. In yet other embodiments, theisolated nucleic acid comprises less than about 10% non-nucleic acidcellular material or cellular protein by mass.

In some embodiments, the array of electrodes comprises a wavy lineconfiguration, wherein the configuration comprises a repeating unitcomprising the shape of a pair of dots connected by linker, wherein thelinker tapers inward toward the midpoint between the pair of dots,wherein the diameters of the dots are the widest points along the lengthof the repeating unit, wherein the edge to edge distance between aparallel set of repeating units is equidistant, or roughly equidistant.In still other embodiments, the array of electrodes comprises apassivation layer with a relative electrical permittivity from about 2.0to about 4.0.

Disclosed herein are methods and devices for analyzing a nucleic acid ina sample comprising: a. applying a sample with a conductivity of greaterthan 100 mS/m to a device, the device comprising an array of electrodescapable of establishing an AC electrokinetic field region, the devicefurther comprising at least two chambers, the sample applied to a firstchamber; b. applying known quantities of a nucleic acid standard to asecond chamber; c. establishing a first AC electrokinetic high fieldregion in the first chamber, the first AC electrokinetic high fieldcapable of isolating larger nanoparticulate molecular targets; d.establishing a second AC electrokinetic low field in the first chamber,the second AC electrokinetic low field capable of concentrating cells ormicron-sized entities that may be present in the sample; e. establishinga third AC electrokinetic high field to the second chamber, the secondAC electrokinetic high field isolating the molecular nucleic acidstandard applied to the second chamber; f. flushing cells andmicron-sized entities that may be present in the sample from the firstchamber; g. detecting bound nucleic acid signal on the array in thefirst chamber and second chamber; h. eluting the bound nucleic acid fromthe first chamber; and i. performing sequencing and/or polymerase chainreaction analysis on the eluted nucleic acid.

Also disclosed herein, in some embodiments, is a method for isolating anucleic acid from a fluid comprising cells, the method comprising: a.applying the fluid to a device, the device comprising an array ofelectrodes capable of establishing an AC electrokinetic field region; b.concentrating a plurality of cells in a first AC electrokinetic fieldregion, wherein the first AC eletrokinetic field region is a firstdielectrophoretic high field region and the conductivity of the fluid isless than 500 mS/m; c. lysing the cells on the array; and d. isolatingnucleic acid in a second AC electrokinetic field region, wherein thesecond AC electrokinetic field is a second dielectrophoretic high fieldregion. In some embodiments, the AC electrokinetic field is producedusing an alternating current having a voltage of 1 volt to 40 voltspeak-peak, and/or a frequency of 5 Hz to 5,000,000 Hz and duty cyclesfrom 5% to 50%. In some embodiments, the conductivity of the fluid isless than 300 mS/m. In some embodiments, the electrodes are selectivelyenergized to provide the first dielectrophoretic high field region andsubsequently or continuously selectively energized to provide the seconddielectrophoretic high field region. In some embodiments, the cells arelysed using a direct current, a chemical lysing agent, an enzymaticlysing agent, heat, pressure, sonic energy, or a combination thereof. Insome embodiments, the method further comprises degradation of residualproteins after cell lysis. In some embodiments, the cells are lysedusing a direct current with a voltage of 1-500 volts, a pulse frequencyof 0.2 to 200 Hz with duty cycles from 10-50%, and a pulse duration of0.01 to 10 seconds applied at least once. In some embodiments, the arrayof electrodes is spin-coated with a hydrogel having a thickness betweenabout 0.1 microns and 1 micron. In some embodiments, the hydrogelcomprises two or more layers of a synthetic polymer. In someembodiments, the hydrogel has a viscosity between about 0.5 cP to about5 cP prior to spin-coating. In some embodiments, the hydrogel has aconductivity between about 0.1 S/m to about 1.0 S/m. In someembodiments, the isolated nucleic acid comprises less than about 10%non-nucleic acid cellular material or cellular protein by mass. In someembodiments, the method is completed in less than 10 minutes. In someembodiments, the array of electrodes comprises a wavy lineconfiguration, wherein the configuration comprises a repeating unitcomprising the shape of a pair of dots connected by linker, wherein thelinker tapers inward toward the midpoint between the pair of dots,wherein the diameters of the dots are the widest points along the lengthof the repeating unit, wherein the edge to edge distance between aparallel set of repeating units is equidistant, or roughly equidistant.In some embodiments, the array of electrodes comprises a passivationlayer with a relative electrical permittivity from about 2.0 to about4.0.

In some embodiments, disclosed herein is a method for isolating anucleic acid from a fluid comprising cells, the method comprising: a.applying the fluid to a device, the device comprising an array ofelectrodes capable of establishing an AC electrokinetic field region; b.concentrating a plurality of cells in a first AC electrokinetic fieldregion, wherein the first AC electrokinetic field region is a firstdielectrophoretic low field region and the conductivity of the fluid isgreater than 300 mS/m; c. isolating nucleic acid in a second ACelectrokinetic field region, wherein the second AC electrokinetic fieldis a second eletrophoretic high field region; and d. flushing cells awayfrom the array. In some embodiments, the AC electrokinetic field isproduced using an alternating current having a voltage of 1 volt to 40volts peak-peak, and/or a frequency of 5 Hz to 5,000,000 Hz and dutycycles from 5% to 50%. In some embodiments, the conductivity of thefluid is greater than 500 mS/m. In some embodiments, the electrodes areselectively energized to provide the first dielectrophoretic high fieldregion and subsequently or continuously selectively energized to providethe second dielectrophoretic high field region. In some embodiments, themethod further comprises degrading residual proteins on the array. Insome embodiments, the residual proteins are degraded by one or more of achemical degradant or an enzymatic degradant. In some embodiments, theresidual proteins are degraded by Proteinase K. In some embodiments, thearray of electrodes is spin-coated with a hydrogel having a thicknessbetween about 0.1 microns and 1 micron. In some embodiments, thehydrogel comprises two or more layers of a synthetic polymer. In someembodiments, the hydrogel has a viscosity between about 0.5 cP to about5 cP prior to spin-coating. In some embodiments, the hydrogel has aconductivity between about 0.1 S/m to about 1.0 S/m. In someembodiments, the isolated nucleic acid comprises less than about 10%non-nucleic acid cellular material or cellular protein by mass. In someembodiments, the method is completed in less than 10 minutes. In someembodiments, the array of electrodes comprises a wavy lineconfiguration, wherein the configuration comprises a repeating unitcomprising the shape of a pair of dots connected by linker, wherein thelinker tapers inward toward the midpoint between the pair of dots,wherein the diameters of the dots are the widest points along the lengthof the repeating unit, wherein the edge to edge distance between aparallel set of repeating units is equidistant, or roughly equidistant.In some embodiments, the array of electrodes comprises a passivationlayer with a relative electrical permittivity from about 2.0 to about4.0.

Disclosed herein, in some embodiments, is a method for isolating anucleic acid from a fluid comprising cells, the method comprising: a.applying the fluid to a device, the device comprising an array ofelectrodes capable of generating an AC electrokinetic field; b.concentrating a plurality of cells in a first AC electrokinetic fieldregion; c. lysing the cells in the first AC electrokinetic field region;and d. isolating the nucleic acid in a second AC electrokinetic fieldregion, wherein the fluid is at a conductivity capable of concentratinga plurality of cells in the first AC electrokinetic field region. Insome embodiments, the first AC electrokinetic region is adielectrophoretic field region, wherein the second AC electrokineticfield region is a dielectrophoretic field region, or a combinationthereof. In some embodiments, the first AC electrokinetic field regionis a first dielectrophoretic low field region and the second ACelectrokinetic field region is a second dielectrophoretic high fieldregion, wherein the conductivity of the fluid is greater than 300 mS/m.In some embodiments, the first AC electrokinetic field region is a firstdielectrophoretic high field region and the second AC electrokineticfield region is a second dielectrophoretic high field region, whereinthe conductivity of the fluid is less than 300 mS/m. In someembodiments, the nucleic acid is concentrated in the second ACelectrokinetic field region. In some embodiments, the method furthercomprises flushing residual material from the array and the isolatednucleic acid. In some embodiments, the method further comprisesdegradation of a residual protein. In some embodiments, the methodfurther comprises flushing degraded proteins from the array and theisolated nucleic acid. In some embodiments, the method further comprisescollecting the nucleic acid. In some embodiments, the first ACelectrokinetic field region is produced by an alternating current. Insome embodiments, the first AC electrokinetic field region is producedusing an alternating current having a voltage of 1 volt to 40 voltspeak-peak; and/or a frequency of 5 Hz to 5,000,000 Hz, and duty cyclesfrom 5% to 50%. In some embodiments, the second AC electrokinetic fieldregion is a different region of the electrode array as the first ACelectrokinetic field region. In some embodiments, the second ACelectrokinetic field region is the same region of the electrode array asthe first AC electrokinetic field region. In some embodiments, thesecond AC electrokinetic field region is produced by an alternatingcurrent. In some embodiments, the second AC electrokinetic field regionis produced using an alternating current having a voltage of 1 volt to50 volts peak-peak; and/or a frequency of 5 Hz to 5,000,000 Hz, and dutycycles from 5% to 50%. In some embodiments, the electrodes areselectively energized to provide the first AC electrokinetic fieldregion and subsequently or continuously selectively energized to providethe second AC electrokinetic field region. In some embodiments, thecells are lysed by applying a direct current to the cells. In someembodiments, the direct current used to lyse the cells has a voltage of1-500 volts; and a duration of 0.01 to 10 seconds applied once or asmultiple pulses. In some embodiments, the direct current used to lysethe cells is a direct current pulse or a plurality of direct currentpulses applied at a frequency suitable for lysing the cells. In someembodiments, the pulse has a frequency of 0.2 to 200 Hz with duty cyclesfrom 10-50%. In some embodiments, the cells are lysed on the deviceusing a direct current, a chemical lysing agent, an enzymatic lysingagent, heat, osmotic pressure, sonic energy, or a combination thereof.In some embodiments, the residual material comprises lysed cellularmaterial. In some embodiments, the lysed cellular material comprisesresidual protein freed from the plurality of cells upon lysis. In someembodiments, the array of electrodes is coated with a hydrogel. In someembodiments, the hydrogel comprises two or more layers of a syntheticpolymer. In some embodiments, the hydrogel is spin-coated onto theelectrodes. In some embodiments, the hydrogel has a viscosity betweenabout 0.5 cP to about 5 cP prior to spin-coating. In some embodiments,the hydrogel has a thickness between about 0.1 microns and 1 micron. Insome embodiments, the hydrogel has a conductivity between about 0.1 S/mto about 1.0 S/m. In some embodiments, the array of electrodes is in adot configuration. In some embodiments, the angle of orientation betweendots is from about 25° to about 60°. In some embodiments, the array ofelectrodes is in a wavy or nonlinear line configuration, wherein theconfiguration comprises a repeating unit comprising the shape of a pairof dots connected by a linker, wherein the dots and linker define theboundaries of the electrode, wherein the linker tapers inward towards orat the midpoint between the pair of dots, wherein the diameters of thedots are the widest points along the length of the repeating unit,wherein the edge to edge distance between a parallel set of repeatingunits is equidistant, or roughly equidistant. In some embodiments, thearray of electrodes comprises a passivation layer with a relativeelectrical permittivity from about 2.0 to about 4.0. In someembodiments, the method further comprises amplifying the isolatednucleic acid by polymerase chain reaction. In some embodiments, thenucleic acid comprises DNA, RNA, or any combination thereof. In someembodiments, the isolated nucleic acid comprises less than about 80%,less than about 70%, less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, less than about10%, less than about 5%, or less than about 2% non-nucleic acid cellularmaterial and/or protein by mass. In some embodiments, the isolatednucleic acid comprises greater than about 99%, greater than about 98%,greater than about 95%, greater than about 90%, greater than about 80%,greater than about 70%, greater than about 60%, greater than about 50%,greater than about 40%, greater than about 30%, greater than about 20%,or greater than about 10% nucleic acid by mass. In some embodiments, themethod is completed in less than about one hour. In some embodiments,centrifugation is not used. In some embodiments, the residual proteinsare degraded by one or more of chemical degradation and enzymaticdegradation. In some embodiments, the residual proteins are degraded byProteinase K. In some embodiments, the residual proteins are degraded byan enzyme, the method further comprising inactivating the enzymefollowing degradation of the proteins. In some embodiments, the enzymeis inactivated by heat (e.g., 50 to 95° C. for 5-15 minutes). In someembodiments, the residual material and the degraded proteins are flushedin separate or concurrent steps. In some embodiments, the isolatednucleic acid is collected by (i) turning off the second ACelectrokinetic field region; and (ii) eluting the nucleic acid from thearray in an eluant. In some embodiments, nucleic acid is isolated in aform suitable for sequencing. In some embodiments, the nucleic acid isisolated in a fragmented form suitable for shotgun-sequencing. In someembodiments, the fluid comprising cells has a low conductivity or a highconductivity. In some embodiments, the fluid comprises a bodily fluid,blood, serum, plasma, urine, saliva, cerebrospinal fluid, body tissue, afood, a beverage, a growth medium, an environmental sample, a liquid,water, clonal cells, or a combination thereof. In some embodiments, thecells comprise clonal cells, pathogen cells, bacteria cells, viruses,plant cells, animal cells, insect cells, and/or combinations thereof. Insome embodiments, the method further comprises sequencing the isolatednucleic acid. In some embodiments, the nucleic acid is sequenced bySanger sequencing, pyrosequencing, ion semiconductor sequencing, polonysequencing, sequencing by ligation, DNA nanoball sequencing, sequencingby ligation, or single molecule sequencing. In some embodiments, themethod further comprises performing a reaction on the DNA (e.g.,fragmentation, restriction digestion, ligation). In some embodiments,the reaction occurs on or near the array or in the device. In someembodiments, the fluid comprising cells comprises no more than 10,000cells.

Disclosed herein, in some embodiments, is a method for isolating anucleic acid from a fluid comprising cells, the method comprising: a.applying the fluid to a device, the device comprising an array ofelectrodes capable of generating an AC electrokinetic field; b.concentrating a plurality of cells in a first AC electrokinetic (e.g.,dielectrophoretic) field region; c. isolating nucleic acid in a secondAC electrokinetic (e.g., dielectrophoretic) field region; and d.flushing cells away, wherein the fluid is at a conductivity capable ofconcentrating a plurality of cells in the first AC electrokinetic fieldregion. In some embodiments, the first AC electrokinetic field region isa dielectrophoretic field region. In some embodiments, the first ACelectrokinetic field region is a dielectrophoretic low field region, andwherein the fluid conductivity is greater than 300 mS/m. In someembodiments, the second AC electrokinetic field region is adielectrophoretic field region. In some embodiments, the method furthercomprises degradation of residual proteins after step (e). In someembodiments, the method further comprises flushing the degraded proteinsfrom the nucleic acid. In some embodiments, the method further comprisescollecting the nucleic acid. In some embodiments, the first ACelectrokinetic field region is produced by an alternating current. Insome embodiments, the first AC electrokinetic field region is producedusing an alternating current having a voltage of 1 volt to 40 voltspeak-peak; and/or a frequency of 5 Hz to 5,000,000 Hz, and duty cyclesfrom 5% to 50%. In some embodiments, the second AC electrokinetic fieldregion is a different region of the electrode array as the first ACelectrokinetic field region. In some embodiments, the second ACelectrokinetic field region is the same region of the electrode array asthe first AC electrokinetic field region. In some embodiments, thesecond AC electrokinetic field region is produced by an alternatingcurrent. In some embodiments, the second AC electrokinetic field regionis a dielectrophoretic high field region. In some embodiments, thesecond AC electrokinetic field region is produced using an alternatingcurrent having a voltage of 1 volt to 50 volts peak-peak; and/or afrequency of 5 Hz to 5,000,000 Hz, and duty cycles from 5% to 50%. Insome embodiments, the electrodes are selectively energized to providethe first AC electrokinetic field region and subsequently orcontinuously selectively energized to provide the second ACelectrokinetic field region. In some embodiments, the array ofelectrodes is coated with a hydrogel. In some embodiments, the hydrogelcomprises two or more layers of a synthetic polymer. In someembodiments, the hydrogel is spin-coated onto the electrodes. In someembodiments, the hydrogel has a viscosity between about 0.5 cP to about5 cP prior to spin-coating. In some embodiments, the hydrogel has athickness between about 0.1 microns and 1 micron. In some embodiments,the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m.In some embodiments, the array of electrodes is in a dot configuration.In some embodiments, the angle of orientation between dots is from about25° to about 60°. In some embodiments, the array of electrodes is in awavy or nonlinear line configuration, wherein the configurationcomprises a repeating unit comprising the shape of a pair of dotsconnected by a linker, wherein the dots and linker define the boundariesof the electrode, wherein the linker tapers inward towards or at themidpoint between the pair of dots, wherein the diameters of the dots arethe widest points along the length of the repeating unit, wherein theedge to edge distance between a parallel set of repeating units isequidistant, or roughly equidistant. In some embodiments, the array ofelectrodes comprises a passivation layer with a relative electricalpermittivity from about 2.0 to about 4.0. In some embodiments, themethod further comprises amplifying the isolated nucleic acid bypolymerase chain reaction. In some embodiments, the nucleic acidcomprises DNA, RNA, or any combination thereof. In some embodiments, theisolated nucleic acid comprises less than about 80%, less than about70%, less than about 60%, less than about 50%, less than about 40%, lessthan about 30%, less than about 20%, less than about 10%, less thanabout 5%, or less than about 2% non-nucleic acid cellular materialand/or protein by mass. In some embodiments, the isolated nucleic acidcomprises greater than about 99%, greater than about 98%, greater thanabout 95%, greater than about 90%, greater than about 80%, greater thanabout 70%, greater than about 60%, greater than about 50%, greater thanabout 40%, greater than about 30%, greater than about 20%, or greaterthan about 10% nucleic acid by mass. In some embodiments, the method iscompleted in less than about one hour. In some embodiments,centrifugation is not used. In some embodiments, the residual proteinsare degraded by one or more of chemical degradation and enzymaticdegradation. In some embodiments, the residual proteins are degraded byProteinase K. In some embodiments, the residual proteins are degraded byan enzyme, the method further comprising inactivating the enzymefollowing degradation of the proteins. In some embodiments, the enzymeis inactivated by heat (e.g., 50 to 95° C. for 5-15 minutes). In someembodiments, the residual material and the degraded proteins are flushedin separate or concurrent steps. In some embodiments, the isolatednucleic acid is collected by (i) turning off the second ACelectrokinetic field region; and (ii) eluting the nucleic acid from thearray in an eluant. In some embodiments, nucleic acid is isolated in aform suitable for sequencing. In some embodiments, the nucleic acid isisolated in a fragmented form suitable for shotgun-sequencing. In someembodiments, the fluid comprising cells has a low conductivity or a highconductivity. In some embodiments, the fluid comprises a bodily fluid,blood, serum, plasma, urine, saliva, cerebrospinal fluid, body tissue, afood, a beverage, a growth medium, an environmental sample, a liquid,water, clonal cells, or a combination thereof. In some embodiments, thecells comprise clonal cells, pathogen cells, bacteria cells, viruses,plant cells, animal cells, insect cells, and/or combinations thereof. Insome embodiments, the method further comprises sequencing the isolatednucleic acid. In some embodiments, the nucleic acid is sequenced bySanger sequencing, pyrosequencing, ion semiconductor sequencing, polonysequencing, sequencing by ligation, DNA nanoball sequencing, sequencingby ligation, or single molecule sequencing. In some embodiments, themethod further comprises performing a reaction on the DNA (e.g.,fragmentation, restriction digestion, ligation). In some embodiments,the reaction occurs on or near the array or in the device. In someembodiments, the fluid comprising cells comprises no more than 10,000cells.

In some embodiments, disclosed herein is a device for isolating anucleic acid from a fluid comprising cells, the device comprising: a. ahousing; b. a heater and/or a reservoir comprising a protein degradationagent; and c. a plurality of alternating current (AC) electrodes withinthe housing, the AC electrodes configured to be selectively energized toestablish AC electrokinetic high field and AC electrokinetic low fieldregions, whereby AC electrokinetic effects provide for concentration ofcells in low field regions of the device. In some embodiments, theplurality of electrodes is configured to be selectively energized toestablish a dielectrophoretic high field and dielectrophoretic low fieldregions. In some embodiments, the array of electrodes is coated with ahydrogel. In some embodiments, the hydrogel comprises two or more layersof a synthetic polymer. In some embodiments, the hydrogel is spin-coatedonto the electrodes. In some embodiments, the hydrogel has a viscositybetween about 0.5 cP to about 5 cP prior to spin-coating. In someembodiments, the hydrogel has a thickness between about 0.1 microns and1 micron. In some embodiments, the hydrogel has a conductivity betweenabout 0.1 S/m to about 1.0 S/m. In some embodiments, the array ofelectrodes is in a dot configuration. In some embodiments, the angle oforientation between dots is from about 25° to about 60°. In someembodiments, the array of electrodes is in a wavy or nonlinear lineconfiguration, wherein the configuration comprises a repeating unitcomprising the shape of a pair of dots connected by a linker, whereinthe dots and linker define the boundaries of the electrode, wherein thelinker tapers inward towards or at the midpoint between the pair ofdots, wherein the diameters of the dots are the widest points along thelength of the repeating unit, wherein the edge to edge distance betweena parallel set of repeating units is equidistant, or roughlyequidistant. In some embodiments, the array of electrodes comprises apassivation layer with a relative electrical permittivity from about 2.0to about 4.0. In some embodiments, the protein degradation agent isProteinase K. In some embodiments, the device further comprises a secondreservoir comprising an eluant.

In some embodiments, disclosed herein is a system for isolating anucleic acid from a fluid comprising cells, the system comprising: a. adevice comprising a plurality of alternating current (AC) electrodes,the AC electrodes configured to be selectively energized to establish ACelectrokinetic high field and AC electrokinetic low field regions,whereby AC electrokinetic effects provide for concentration of cells inhigh field regions of the device, wherein the configuration comprises arepeating unit comprising the shape of a pair of dots connected by alinker, wherein the dots and linker define the boundaries of theelectrode, wherein the linker tapers inward towards or at the midpointbetween the pair of dots, wherein the diameters of the dots are thewidest points along the length of the repeating unit, wherein the edgeto edge distance between a parallel set of repeating units isequidistant, or roughly equidistant; and b. a module capable ofsequencing DNA by Sanger sequencing or next generation sequencingmethods; c. a software program capable of controlling the devicecomprising a plurality of AC electrodes, the module capable ofsequencing DNA or a combination thereof; and d. a fluid comprisingcells. In some embodiments, the plurality of electrodes is configured tobe selectively energized to establish a dielectrophoretic high field anddielectrophoretic low field regions.

Disclosed herein, in some embodiments, is a device comprising: a. aplurality of alternating current (AC) electrodes, the AC electrodesconfigured to be selectively energized to establish AC electrokinetichigh field and AC electrokinetic low field regions, wherein the array ofelectrodes is in a wavy or nonlinear line configuration, wherein theconfiguration comprises a repeating unit comprising the shape of a pairof dots connected by a linker, wherein the dots and linker define theboundaries of the electrode, wherein the linker tapers inward towards orat the midpoint between the pair of dots, wherein the diameters of thedots are the widest points along the length of the repeating unit,wherein the edge to edge distance between a parallel set of repeatingunits is equidistant, or roughly equidistant; and b. a module capable ofthermocycling and amplifying nucleic acids. In some embodiments, theplurality of electrodes is configured to be selectively energized toestablish a dielectrophoretic high field and dielectrophoretic low fieldregions. In some embodiments, the device is capable of isolating nucleicacids from a fluid comprising cells and performing amplification of theisolated nucleic acids. In some embodiments, the isolated nucleic acidis DNA or mRNA. In some embodiments, nucleic acid is isolated andamplification is performed in a single chamber. In some embodiments,nucleic acid is isolated and amplification is performed in multipleregions of a single chamber. In some embodiments, the device furthercomprises using at least one of an elution tube, a chamber and areservoir to perform amplification. In some embodiments, amplificationof the nucleic acid is polymerase chain reaction (PCR)-based. In someembodiments, amplification of the nucleic acid is performed in aserpentine microchannel comprising a plurality of temperature zones. Insome embodiments, amplification is performed in aqueous dropletsentrapped in immiscible fluids (i.e., digital PCR). In some embodiments,the thermocycling comprises convection. In some embodiments, the devicecomprises a surface contacting or proximal to the electrodes, whereinthe surface is functionalized with biological ligands that are capableof selectively capturing biomolecules. In some embodiments, the array ofelectrodes is coated with a hydrogel. In some embodiments, the hydrogelcomprises two or more layers of a synthetic polymer. In someembodiments, the hydrogel is spin-coated onto the electrodes. In someembodiments, the hydrogel has a viscosity between about 0.5 cP to about5 cP prior to spin-coating. In some embodiments, the hydrogel has athickness between about 0.1 microns and 1 micron. In some embodiments,the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m.In some embodiments, the array of electrodes comprises a passivationlayer with a relative electrical permittivity from about 2.0 to about4.0. In some embodiments, the surface selectively captures biomoleculesby: a. nucleic acid hybridization; b. antibody-antigen interactions; c.biotin-avidin interactions; d. ionic or electrostatic interactions; ore. any combination thereof. In some embodiments, the surface isfunctionalized to minimize and/or inhibit nonspecific bindinginteractions by: a. polymers (e.g., polyethylene glycol PEG); b. ionicor electrostatic interactions; c. surfactants; or d. any combinationthereof. In some embodiments, the device comprises a plurality ofmicroelectrode devices oriented (a) flat side by side, (b) facingvertically, or (c) facing horizontally. In some embodiments, the devicecomprises a module capable of performing Sanger sequencing. In someembodiments, the module capable of performing Sanger sequencingcomprises a module capable of capillary electrophoresis, a modulecapable of multi-color fluorescence detection, or a combination thereof.

Disclosed herein, in some embodiments, is a device comprising: a. aplurality of alternating current (AC) electrodes, the AC electrodesconfigured to be selectively energized to establish AC electrokinetichigh field and AC electrokinetic low field regions, wherein the array ofelectrodes is in a wavy or nonlinear line configuration, wherein theconfiguration comprises a repeating unit comprising the shape of a pairof dots connected by a linker, wherein the dots and linker define theboundaries of the electrode, wherein the linker tapers inward towards orat the midpoint between the pair of dots, wherein the diameters of thedots are the widest points along the length of the repeating unit,wherein the edge to edge distance between a parallel set of repeatingunits is equidistant, or roughly equidistant; and b. a module capable ofperforming sequencing. In some embodiments, the plurality of electrodesis configured to be selectively energized to establish adielectrophoretic high field and dielectrophoretic low field regions. Insome embodiments, the device comprises a surface contacting or proximalto the electrodes, wherein the surface is functionalized with biologicalligands that are capable of selectively capturing biomolecules. In someembodiments, the array of electrodes is coated with a hydrogel. In someembodiments, the hydrogel comprises two or more layers of a syntheticpolymer. In some embodiments, the hydrogel is spin-coated onto theelectrodes. In some embodiments, the hydrogel has a viscosity betweenabout 0.5 cP to about 5 cP prior to spin-coating. In some embodiments,the hydrogel has a thickness between about 0.1 microns and 1 micron. Insome embodiments, the hydrogel has a conductivity between about 0.1 S/mto about 1.0 S/m. In some embodiments, the array of electrodes comprisesa passivation layer with a relative electrical permittivity from about2.0 to about 4.0. In some embodiments, the surface selectively capturesbiomolecules by: a. nucleic acid hybridization; b. antibody-antigeninteractions; c. biotin-avidin interactions; d. ionic or electrostaticinteractions; or e. any combination thereof. In some embodiments, thesurface is functionalized to minimize and/or inhibit nonspecific bindinginteractions by: a. polymers (e.g., polyethylene glycol PEG); b. ionicor electrostatic interactions; c. surfactants; or d. any combinationthereof. In some embodiments, the device comprises a plurality ofmicroelectrode devices oriented (a) flat side by side, (b) facingvertically, or (c) facing horizontally. In some embodiments, the devicecomprises a module capable of performing next generation sequencing. Insome embodiments, the module capable of performing next-generationsequencing is capable of performing pyrosequencing, ion semiconductorsequencing, polony sequencing, sequencing by ligation, DNA nanoballsequencing, or single molecule sequencing.

Disclosed herein, in some embodiments, is a method of isolating anucleic acid from a fluid comprising cells, comprising a) performing amethod disclosed herein; b) performing PCR amplification on the nucleicacid, or a cDNA version of the nucleic acid, to produce a PCR product;c) isolating the PCR product in a third AC electrokinetic region; d)performing Sanger chain termination reactions on the PCR product toproduce a sequencing product of the nucleic acid; and e) performingelectrophoretic separation of the sequencing product of the nucleicacid. In some embodiments, the third AC electrokinetic region is adielectrophoretic field region. In some embodiments, the third ACelectrokinetic region is a dielectrophoretic high field region. In someembodiments, the array of electrodes is in a wavy or nonlinear lineconfiguration, wherein the configuration comprises a repeating unitcomprising the shape of a pair of dots connected by a linker, whereinthe dots and linker define the boundaries of the electrode, wherein thelinker tapers inward towards or at the midpoint between the pair ofdots, wherein the diameters of the dots are the widest points along thelength of the repeating unit, wherein the edge to edge distance betweena parallel set of repeating units is equidistant, or roughlyequidistant. In some embodiments, the electrophoretic separation of thesequencing product of the nucleic acid is capillary electrophoresis. Insome embodiments, the method further comprises the use of multicolorfluorescence detection to analyze the sequencing product of the nucleicacid. In some embodiments, all steps are performed on a single chip. Insome embodiments, the fluid comprising cells comprises no more than10,000 cells.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a top view (A), a bottom view (B) and a cross-sectionalview (C) of an exemplary device.

FIG. 2 shows the electrodes associated with various amounts of genomicDNA.

FIG. 3 shows isolation of green fluorescent E. coli on an array. Panel(A) shows a bright field view. Panel (B) shows a green fluorescent viewof the electrodes before DEP activation. Panel (C) shows E. coli on theelectrodes after one minute at 10 kHz, 20 Vp-p in 1×TBE buffer. Panel(D) shows E. coli on the electrodes after one minute at 1 MHz, 20 Vp-pin 1×TBE buffer.

FIG. 4 shows a comparison between the methods of the present invention(top right panel) and the Epicentre™ WaterMaster™ DNA purificationprocedure (top left panel). The pie charts are the distribution of10,000 Illumina™ sequencing reads BLAST searched against the MEGAN™database. As shown, a similar percentage of sequencing reads originatedfrom E. coli sequence for both methods. The table in the lower panelshows Sequencing coverage and quality of E. Coli run through the chipand compared to a control run outside the chip according tomanufacturer's protocol.

FIG. 5 shows an exemplary method for isolating nucleic acids from cells.

FIG. 6 shows an exemplary method for isolating extra-cellular nucleicacids from a fluid comprising cells.

FIG. 7 exemplifies ACE (AC Electrokinetic) forces that result due to themethods and devices disclosed herein. Using the relationship betweenforces on particles due to Dielectrophoresis (DEP), AC Electrothermal(ACET) flow and AC Electroosmosis, (ACEO), in some embodiments, sizecut-offs are used for nucleic acid isolation and purification. Isolationrelies on flow vortices that will brings nucleic acids closer to anelectrode edge due to ACET and ACED depending on fluid conductivity, ADEP trap holds onto particles once they are at the trap site, dependingon the effective Stokes radius.

FIG. 8 exemplifies a wavy electrode configuration, as disclosed herein.The edge to edge distance between electrodes is generally equidistantthroughout. A wavy electrode configuration maximizes electrode surfacearea while maintaining alternating non-uniform electric field to induceACE gradient to enable DEP, ACEO, ACET, and other ACE forces.

FIG. 9 exemplifies how the E-field gradient at a dielectric layer cornerbased on silicon nitride thickness. Lower K and lower thickness resultedin higher E-field gradient (bending) at a dielectric layer corner.

FIG. 10 exemplifies DNA capture on an electrode with a vapor depositedhydrogel layer. Vapor phase coatings of activated monomers form uniformthin film coatings on a variety of substrates. Hydrogels such as pHEMAwere deposited in various thickness (100, 200, 300, 400 nm) andcrosslinking (5, 25, 40%) density on electrode chips by GVD Corporation(Cambridge, Mass.). The hydrogel films were tested using a standard ACEprotocol (no pretreatment, 7Vp-p, 10 KHz, 2 minutes, 0.5×PBS, 500 ng/mlgDNA labeled with SYBR® Green 1). Fluorescence on the electrodes wascaptured by imaging. The 100 nm thickness, 5% crosslink gel device wasfound to have strong DNA capture. Optionally, the process could beoptimized by changing the deposition rate or anchoring growth to thesurface of the microelectrode array (i.e., to the passivation layer andexposed electrodes), using an adhesion promote such as a silanederivative.

FIG. 11 shows a picture of a two-chamber fluidic cartridge showing thelayout for the unknown (U) and known (K) chambers.

FIG. 12 shows a fluorescent image of YOYO®-1 labelled circulatingcell-free DNA captured on electrodes. Region-of-Interest (ROI)segmentation enables rapid processing conversion from image toquantitative score.

FIG. 13 illustrates the linear calibration for relative fluorescentunits vs DNA concentration based on ROI segmentation of captured DNA onACE electrodes.

FIG. 14 shows the compatibility of the ACE fluidic wash solution withthe downstream PCR mutation detection assays. DNA samples from the celllines H1975, RKO, OCI-AML3 and HEL 92.1.7 were diluted in either H₂O orACE wash solution and used as positive controls for EGFR T790, BRAFV600, NPM1a and JAK2 617V assays, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are methods, devices and systems suitable for isolatingor separating particles or molecules from a fluid composition. Inspecific embodiments, provided herein are methods, devices and systemsfor isolating or separating a nucleic acid from a fluid comprising cellsor other particulate material. In some aspects, the methods, devices andsystems may allow for rapid separation of particles and molecules in afluid composition. In other aspects, the methods, devices and systemsmay allow for rapid isolation of molecules from particles in a fluidcomposition. In various aspects, the methods, devices and systems mayallow for a rapid procedure that requires a minimal amount of materialand/or results in high purity DNA isolated from complex fluids such asblood or environmental samples.

Provided in certain embodiments herein are methods, devices and systemsfor isolating or separating particles or molecules from a fluidcomposition, the methods, devices, and systems comprising applying thefluid to a device comprising an array of electrodes and being capable ofgenerating AC electrokinetic forces (e.g., when the array of electrodesare energized). In some embodiments, the dielectrophoretic field, is acomponent of AC electrokinetic force effects. In other embodiments, thecomponent of AC electrokinetic force effects is AC electroosmosis or ACelectrothermal effects. In some embodiments the AC electrokinetic force,including dielectrophoretic fields, comprises high-field regions(positive DEP, i.e. area where there is a strong concentration ofelectric field lines due to a non-uniform electric field) and/orlow-field regions (negative DEP, i.e. area where there is a weakconcentration of electric field lines due to a non-uniform electricfield).

In specific instances, the particles or molecules (e.g., nucleic acid)are isolated (e.g., isolated or separated from cells) in a field region(e.g., a high field region) of the dielectrophoretic field. In someembodiments, the method, device, or system further includes one or moreof the following steps: concentrating cells of interest in a firstdielectrophoretic field region (e.g., a high field DEP region), lysingcells in the first dielectrophoretic field region, and/or concentratingnucleic acid in a first or second dielectrophoretic field region. Inother embodiments, the method, device, or system includes one or more ofthe following steps: concentrating cells in a first dielectrophoreticfield region (e.g., a low field DEP region), concentrating nucleic acidin a second dielectrophoretic field region (e.g., a high field DEPregion), and washing away the cells and residual material. The methodalso optionally includes devices and/or systems capable of performingone or more of the following steps: washing or otherwise removingresidual (e.g., cellular) material from the nucleic acid (e.g., rinsingthe array with water or buffer while the nucleic acid is concentratedand maintained within a high field DEP region of the array), degradingresidual proteins (e.g., residual proteins from lysed cells and/or othersources, such degradation occurring according to any suitable mechanism,such as with heat, a protease, or a chemical), flushing degradedproteins from the nucleic acid, and collecting the nucleic acid. In someembodiments, the result of the methods, operation of the devices, andoperation of the systems described herein is an isolated nucleic acid,optionally of suitable quantity and purity for DNA sequencing.

In some instances, it is advantageous that the methods described hereinare performed in a short amount of time, the devices are operated in ashort amount of time, and the systems are operated in a short amount oftime. In some embodiments, the period of time is short with reference tothe “procedure time” measured from the time between adding the fluid tothe device and obtaining isolated nucleic acid. In some embodiments, theprocedure time is less than 3 hours, less than 2 hours, less than 1hour, less than 30 minutes, less than 20 minutes, less than 10 minutes,or less than 5 minutes.

In another aspect, the period of time is short with reference to the“hands-on time” measured as the cumulative amount of time that a personmust attend to the procedure from the time between adding the fluid tothe device and obtaining isolated nucleic acid. In some embodiments, thehands-on time is less than 20 minutes, less than 10 minutes, less than 5minute, less than 1 minute, or less than 30 seconds.

In some instances, it is advantageous that the devices described hereincomprise a single vessel, the systems described herein comprise a devicecomprising a single vessel and the methods described herein can beperformed in a single vessel, e.g., in a dielectrophoretic device asdescribed herein. In some aspects, such a single-vessel embodimentminimizes the number of fluid handling steps and/or is performed in ashort amount of time. In some instances, the present methods, devicesand systems are contrasted with methods, devices and systems that useone or more centrifugation steps and/or medium exchanges. In someinstances, centrifugation increases the amount of hands-on time requiredto isolate nucleic acids. In another aspect, the single-vessel procedureor device isolates nucleic acids using a minimal amount of consumablereagents.

Devices and Systems

In some embodiments, described herein are devices for collecting anucleic acid from a fluid. In one aspect, described herein are devicesfor collecting a nucleic acid from a fluid comprising cells or otherparticulate material. In other aspects, the devices disclosed herein arecapable of collecting and/or isolating nucleic acid from a fluidcomprising cellular or protein material. In other instances, the devicesdisclosed herein are capable of collecting and/or isolating nucleic acidfrom cellular material.

In some embodiments, disclosed herein is a device for isolating anucleic acid from a fluid comprising cells or other particulatematerial, the device comprising: a. a housing; b. a heater or thermalsource and/or a reservoir comprising a protein degradation agent; and c.a plurality of alternating current (AC) electrodes within the housing,the AC electrodes configured to be selectively energized to establish ACelectrokinetic high field and AC electrokinetic low field regions,whereby AC electrokinetic effects provide for concentration of cells inlow field regions of the device. In some embodiments, the plurality ofelectrodes is configured to be selectively energized to establish adielectrophoretic high field and dielectrophoretic low field regions. Insome embodiments, the protein degradation agent is a protease. In someembodiments, the protein degradation agent is Proteinase K. In someembodiments, the device further comprises a second reservoir comprisingan eluant.

In some embodiments, disclosed herein is a device comprising: a. aplurality of alternating current (AC) electrodes, the AC electrodesconfigured to be selectively energized to establish AC electrokinetichigh field and AC electrokinetic low field regions; and b. a modulecapable of thermocycling and performing PCR or other enzymaticreactions. In some embodiments, the plurality of electrodes isconfigured to be selectively energized to establish a dielectrophoretichigh field and dielectrophoretic low field regions. In some embodiments,the device is capable of isolating DNA from a fluid comprising cells andperforming PCR amplification or other enzymatic reactions. In someembodiments, DNA is isolated and PCR or other enzymatic reaction isperformed in a single chamber. In some embodiments, DNA is isolated andPCR or other enzymatic reaction is performed in multiple regions of asingle chamber. In some embodiments, DNA is isolated and PCR or otherenzymatic reaction is performed in multiple chambers.

In some embodiments, the device further comprises at least one of anelution tube, a chamber and a reservoir to perform PCR amplification orother enzymatic reaction. In some embodiments, PCR amplification orother enzymatic reaction is performed in a serpentine microchannelcomprising a plurality of temperature zones. In some embodiments, PCRamplification or other enzymatic reaction is performed in aqueousdroplets entrapped in immiscible fluids (i.e., digital PCR). In someembodiments, the thermocycling comprises convection. In someembodiments, the device comprises a surface contacting or proximal tothe electrodes, wherein the surface is functionalized with biologicalligands that are capable of selectively capturing biomolecules.

In some embodiments, disclosed herein is a system for isolating anucleic acid from a fluid comprising cells or other particulatematerial, the system comprising: a. a device comprising a plurality ofalternating current (AC) electrodes, the AC electrodes configured to beselectively energized to establish AC electrokinetic high field and ACelectrokinetic low field regions, whereby AC electrokinetic effectsprovide for concentration of cells in high field regions of the device;and b. a sequencer, thermocycler or other device for performingenzymatic reactions on isolated or collected nucleic acid. In someembodiments, the plurality of electrodes is configured to be selectivelyenergized to establish a dielectrophoretic high field anddielectrophoretic low field regions.

In various embodiments, DEP fields are created or capable of beingcreated by selectively energizing an array of electrodes as describedherein. The electrodes are optionally made of any suitable materialresistant to corrosion, including metals, such as noble metals (e.g.platinum, platinum iridium alloy, palladium, gold, and the like). Invarious embodiments, electrodes are of any suitable size, of anysuitable orientation, of any suitable spacing, energized or capable ofbeing energized in any suitable manner, and the like such that suitableDEP and/or other electrokinetic fields are produced.

In some embodiments described herein are methods, devices and systems inwhich the electrodes are placed into separate chambers and positive DEPregions and negative DEP regions are created within an inner chamber bypassage of the AC DEP field through pore or hole structures. Variousgeometries are used to form the desired positive DEP (high field)regions and DEP negative (low field) regions for carrying cellular,microparticle, nanoparticle, and nucleic acid separations. In someembodiments, pore or hole structures contain (or are filled with) porousmaterial (hydrogels) or are covered with porous membrane structures. Insome embodiments, by segregating the electrodes into separate chambers,such pore/hole structure DEP devices reduce electrochemistry effects,heating, or chaotic fluidic movement from occurring in the innerseparation chamber during the DEP process.

In one aspect, described herein is a device comprising electrodes,wherein the electrodes are placed into separate chambers and DEP fieldsare created within an inner chamber by passage through pore structures.The exemplary device includes a plurality of electrodes andelectrode-containing chambers within a housing. A controller of thedevice independently controls the electrodes, as described further inPCT patent publication WO 2009/146143 A2, which is incorporated hereinfor such disclosure.

In some embodiments, chambered devices are created with a variety ofpore and/or hole structures (nanoscale, microscale and even macroscale)and contain membranes, gels or filtering materials which control,confine or prevent cells, nanoparticles or other entities from diffusingor being transported into the inner chambers while the AC/DC electricfields, solute molecules, buffer and other small molecules can passthrough the chambers.

In various embodiments, a variety of configurations for the devices arepossible. For example, a device comprising a larger array of electrodes,for example in a square or rectangular pattern configured to create arepeating non-uniform electric field to enable AC electrokinetics. Forillustrative purposes only, a suitable electrode array may include, butis not limited to, a 10×10 electrode configuration, a 50×50 electrodeconfiguration, a 10×100 electrode configuration, 20×100 electrodeconfiguration, or a 20×80 electrode configuration.

Such devices include, but are not limited to, multiplexed electrode andchambered devices, devices that allow reconfigurable electric fieldpatterns to be created, devices that combine DC electrophoretic andfluidic processes; sample preparation devices, sample preparation,enzymatic manipulation of isolated nucleic acid molecules and diagnosticdevices that include subsequent detection and analysis, lab-on-chipdevices, point-of-care and other clinical diagnostic systems orversions.

In some embodiments, a planar platinum electrode array device comprisesa housing through which a sample fluid flows. In some embodiments, fluidflows from an inlet end to an outlet end, optionally comprising alateral analyte outlet. The exemplary device includes multiple ACelectrodes.

In some embodiments, the sample consists of a combination ofmicron-sized entities or cells, larger nanoparticulates and smallernanoparticulates or biomolecules. In some embodiments, the micron-sizedentities may comprise blood cells, platelets, bacteria and the like. Insome embodiments, larger nanoparticulates comprise particulates in therange of about 10 nm and about 900 nm effective stokes diameter, and maycomprise exosomes, high mw nucleic acids, including high mw DNA,oligo-nucleosome complexes, aggregated proteins, vesicle bound DNA, cellmembrane fragments and cellular debris dispersed in the sample. In someembodiments, smaller nanoparticulates (<10 nm effective stokes diameter)comprise proteins such as immunoglobulins, human serum albumin,fibrinogen and other plasma proteins, smaller apoptotic DNA, and freeions.

In some embodiments, the AC electrokinetic field regions disclosedherein are capable of selectively isolating target particulates,including micron-sized entities, larger nanoparticulates and/or smallernanoparticulates. In some embodiments, the AC electrokinetic fieldregions disclosed herein are capable of selectively isolating targetparticulates, including micron-sized entities, larger nanoparticulatesand/or smaller nanoparticulates in complex biological or environmentalsamples. The target particulates are isolated in different field regionsat or near the surface of the array, allowing non-target particulates orparticulates that are not isolated at or near the surface of the arrayto be flushed from the array or cartridge.

In some embodiments, the planar electrode array device is a 60×20electrode array that is optionally sectioned into three 20×20 arraysthat can be separately controlled but operated simultaneously. Theoptional auxiliary DC electrodes can be switched on to positive charge,while the optional DC electrodes are switched on to negative charge forelectrophoretic purposes. In some instances, each of the controlled ACand DC systems is used in both a continuous and/or pulsed manner (e.g.,each can be pulsed on and off at relatively short time intervals) invarious embodiments. The optional planar electrode arrays along thesides of the sample flow, when over-layered with nanoporous material(e.g., a hydrogel of synthetic polymer), are optionally used to generateDC electrophoretic forces as well as AC DEP. Additionally,microelectrophoretic separation processes is optionally carried outwithin the nanopore layers using planar electrodes in the array and/orauxiliary electrodes in the x-y-z dimensions.

In various embodiments these methods, devices and systems are operatedin the AC frequency range of from 1,000 Hz to 100 MHz, at voltages whichcould range from approximately 1 volt to 2000 volts pk-pk; at DCvoltages from 1 volt to 1000 volts, at flow rates of from 10 microlitersper minute to 10 milliliter per minute, and in temperature ranges from1° C. to 120° C. In some embodiments, the methods, devices and systemsare operated in AC frequency ranges of from about 3 to about 15 kHz. Insome embodiments, the methods, devices, and systems are operated atvoltages of from 5-25 volts pk-pk. In some embodiments, the methods,devices and systems are operated at voltages of from about 1 to about 50volts/cm. In some embodiments, the methods, devices and systems areoperated at DC voltages of from about 1 to about 5 volts. In someembodiments, the methods, devices and systems are operated at a flowrate of from about 10 microliters to about 500 microliters per minute.In some embodiments, the methods, devices and systems are operated intemperature ranges of from about 20° C. to about 60° C. In someembodiments, the methods, devices and systems are operated in ACfrequency ranges of from 1,000 Hz to 10 MHz. In some embodiments, themethods, devices and systems are operated in AC frequency ranges of from1,000 Hz to 1 MHz. In some embodiments, the methods, devices and systemsare operated in AC frequency ranges of from 1,000 Hz to 100 kHz. In someembodiments, the methods, devices and systems are operated in ACfrequency ranges of from 1,000 Hz to 10 kHz. In some embodiments, themethods, devices and systems are operated in AC frequency ranges of from10 kHz to 100 kHz. In some embodiments, the methods, devices and systemsare operated in AC frequency ranges of from 100 kHz to 1 MHz. In someembodiments, the methods, devices and systems are operated at voltagesfrom approximately 1 volt to 1500 volts pk-pk. In some embodiments, themethods, devices and systems are operated at voltages from approximately1 volt to 1500 volts pk-pk. In some embodiments, the methods, devicesand systems are operated at voltages from approximately 1 volt to 1000volts pk-pk. In some embodiments, the methods, devices and systems areoperated at voltages from approximately 1 volt to 500 volts pk-pk. Insome embodiments, the methods, devices and systems are operated atvoltages from approximately 1 volt to 250 volts pk-pk. In someembodiments, the methods, devices and systems are operated at voltagesfrom approximately 1 volt to 100 volts pk-pk. In some embodiments, themethods, devices and systems are operated at voltages from approximately1 volt to 50 volts pk-pk. In some embodiments, the methods, devices andsystems are operated at DC voltages from 1 volt to 1000 volts. In someembodiments, the methods, devices and systems are operated at DCvoltages from 1 volt to 500 volts. In some embodiments, the methods,devices and systems are operated at DC voltages from 1 volt to 250volts. In some embodiments, the methods, devices and systems areoperated at DC voltages from 1 volt to 100 volts. In some embodiments,the methods, devices and systems are operated at DC voltages from 1 voltto 50 volts. In some embodiments, the methods, devices, and systems areoperated at flow rates of from 10 microliters per minute to 1 ml perminute. In some embodiments, the methods, devices, and systems areoperated at flow rates of from 10 microliters per minute to 500microliters per minute. In some embodiments, the methods, devices, andsystems are operated at flow rates of from 10 microliters per minute to250 microliters per minute. In some embodiments, the methods, devices,and systems are operated at flow rates of from 10 microliters per minuteto 100 microliters per minute. In some embodiments, the methods,devices, and systems are operated in temperature ranges from 1° C. to100° C. In some embodiments, the methods, devices, and systems areoperated in temperature ranges from 20° C. to 95° C. In someembodiments, the methods, devices, and systems are operated intemperature ranges from 25° C. to 100° C. In some embodiments, themethods, devices, and systems are operated at room temperature.

In some embodiments, the controller independently controls each of theelectrodes. In some embodiments, the controller is externally connectedto the device such as by a socket and plug connection, or is integratedwith the device housing.

Also described herein are scaled sectioned (x-y dimensional) arrays ofrobust electrodes and strategically placed (x-y-z dimensional)arrangements of auxiliary electrodes that combine DEP, electrophoretic,and fluidic forces, and use thereof. In some embodiments, clinicallyrelevant volumes of blood, serum, plasma, or other samples are moredirectly analyzed under higher ionic strength and/or conductanceconditions. Described herein is the overlaying of robust electrodestructures (e.g. platinum, palladium, gold, etc.) with one or moreporous layers of materials (natural or synthetic porous hydrogels,membranes, controlled nanopore materials, and thin dielectric layeredmaterials) to reduce the effects of any electrochemistry (electrolysis)reactions, heating, and chaotic fluid movement that may occur on or nearthe electrodes, and still allow the effective separation of cells,bacteria, virus, nanoparticles, DNA, and other biomolecules to becarried out. In some embodiments, in addition to using AC frequencycross-over points to achieve higher resolution separations, on-device(on-array) DC microelectrophoresis is used for secondary separations.For example, the separation of DNA nanoparticulates (20-50 kb), highmolecular weight DNA (5-20 kb), intermediate molecular weight DNA (1-5kb), and lower molecular weight DNA (0.1-1 kb) fragments may beaccomplished through DC microelectrophoresis on the array. In someembodiments, the device is sub-sectioned, optionally for purposes ofconcurrent separations of different blood cells, bacteria and virus, andDNA carried out simultaneously on such a device.

In some embodiments, the device comprises a housing and a heater orthermal source and/or a reservoir comprising a protein degradationagent. In some embodiments, the heater or thermal source is capable ofincreasing the temperature of the fluid to a desired temperature (e.g.,to a temperature suitable for degrading proteins, about 30° C., 40° C.,50° C., 60° C., 70° C., or the like). In some embodiments, the heater orthermal source is suitable for operation as a PCR thermocycler. IN otherembodiments, the heater or thermal source is used to maintain a constanttemperature (isothermal conditions). In some embodiments, the proteindegradation agent is a protease. In other embodiments, the proteindegradation agent is Proteinase K and the heater or thermal source isused to inactivate the protein degradation agent.

In some embodiments, the device also comprises a plurality ofalternating current (AC) electrodes within the housing, the ACelectrodes capable of being configured to be selectively energized toestablish dielectrophoretic (DEP) high field and dielectrophoretic (DEP)low field regions, whereby AC electrokinetic effects provide forconcentration of cells in low field regions of the device. In someembodiments, the electrodes are selectively energized to provide thefirst AC electrokinetic field region and subsequently or continuouslyselectively energized to provide the second AC electrokinetic fieldregion. For example, further description of the electrodes and theconcentration of cells in DEP fields is found in PCT patent publicationWO 2009/146143 A2, which is incorporated herein for such disclosure.

In some embodiments, the device comprises a second reservoir comprisingan eluant. The eluant is any fluid suitable for eluting the isolatednucleic acid from the device. In some instances the eluant is water or abuffer. In some instances, the eluant comprises reagents required for aDNA sequencing method.

Also provided herein are systems and devices comprising a plurality ofalternating current (AC) electrodes, the AC electrodes configured to beselectively energized to establish dielectrophoretic (DEP) high fieldand dielectrophoretic (DEP) low field regions. In some instances, ACelectrokinetic effects provide for concentration of cells in low fieldregions and/or concentration (or collection or isolation) of molecules(e.g., macromolecules, such as nucleic acid) in high field regions ofthe DEP field.

Also provided herein are systems and devices comprising a plurality ofdirect current (DC) electrodes. In some embodiments, the plurality of DCelectrodes comprises at least two rectangular electrodes, spreadthroughout the array. In some embodiments, the electrodes are located atthe edges of the array. In some embodiments, DC electrodes areinterspersed between AC electrodes.

In some embodiments, a system or device described herein comprises ameans for manipulating nucleic acid. In some embodiments, a system ordevice described herein includes a means of performing enzymaticreactions. In other embodiments, a system or device described hereinincludes a means of performing polymerase chain reaction, isothermalamplification, ligation reactions, restriction analysis, nucleic acidcloning, transcription or translation assays, or other enzymatic-basedmolecular biology assay. In yet other embodiments, a system or devicedescribed herein includes a means of performing Quantitative Real TimePCR, including of nuclear or mitochondrial DNA, enzyme-linkedimmunosorbent assays (ELISA), direct SYBR gold assays, direct PicoGreenassays, loss of heterozygosity (LOH) of microsatellite markers,optionally followed by electrophoresis analysis, including but notlimited to capillary electrophoresis analysis, sequencing and/orcloning, including next generation sequencing, methylation analysis,including but not limited to modified semi-nested or nested methylationspecific PCR, DNA specific PCR (MSP), quantification of minute amountsof DNA after bisulfitome amplification (qMAMBRA), as well as methylationon beads, mass-based analysis, including but not limited to MALDI-ToF(matrix-assisted laser desorption/ionization time of flight analysis,optionally in combination with PCR, and digital PCR.

In some embodiments, a system or device described herein comprises anucleic acid sequencer. The sequencer is optionally any suitable DNAsequencing device including but not limited to a Sanger sequencer,gyro-sequencer, ion semiconductor sequencer, polony sequencer,sequencing by ligation device, DNA nanoball sequencing device,sequencing by ligation device, or single molecule sequencing device.

In some embodiments, a system or device described herein is capable ofmaintaining a constant temperature. In some embodiments, a system ordevice described herein is capable of cooling the array or chamber. Insome embodiments, a system or device described herein is capable ofheating the array or chamber. In some embodiments, a system or devicedescribed herein comprises a thermocycler. In some embodiments, thedevices disclosed herein comprises a localized temperature controlelement. In some embodiments, the devices disclosed herein are capableof both sensing and controlling temperature.

In some embodiments, the devices further comprise heating or thermalelements. In some embodiments, a heating or thermal element is localizedunderneath an electrode. In some embodiments, the heating or thermalelements comprise a metal. In some embodiments, the heating or thermalelements comprise tantalum, aluminum, tungsten, or a combinationthereof. Generally, the temperature achieved by a heating or thermalelement is proportional to the current running through it. In someembodiments, the devices disclosed herein comprise localized coolingelements. In some embodiments, heat resistant elements are placeddirectly under the exposed electrode array. In some embodiments, thedevices disclosed herein are capable of achieving and maintaining atemperature between about 20° C. and about 120° C. In some embodiments,the devices disclosed herein are capable of achieving and maintaining atemperature between about 30° C. and about 100° C. In other embodiments,the devices disclosed herein are capable of achieving and maintaining atemperature between about 20° C. and about 95° C. In some embodiments,the devices disclosed herein are capable of achieving and maintaining atemperature between about 25° C. and about 90° C., between about 25° C.and about 85° C., between about 25° C. and about 75° C., between about25° C. and about 65° C. or between about 25° C. and about 55° C. In someembodiments, the devices disclosed herein are capable of achieving andmaintaining a temperature of about 20° C., about 30° C., about 40° C.,about 50° C., about 60° C., about 70° C., about 80° C., about 90° C.,about 100° C., about 110° C. or about 120° C.

Electrodes

The plurality of alternating current electrodes are optionallyconfigured in any manner suitable for the separation processes describedherein. For example, further description of the system or deviceincluding electrodes and/or concentration of cells in DEP fields isfound in PCT patent publication WO 2009/146143, which is incorporatedherein for such disclosure.

In some embodiments, the electrodes disclosed herein can comprise anysuitable metal. In some embodiments, the electrodes can include but arenot limited to: aluminum, copper, carbon, iron, silver, gold, palladium,platinum, iridium, platinum iridium alloy, ruthenium, rhodium, osmium,tantalum, titanium, tungsten, polysilicon, and indium tin oxide, orcombinations thereof, as well as silicide materials such as platinumsilicide, titanium silicide, gold silicide, or tungsten silicide. Insome embodiments, the electrodes can comprise a conductive ink capableof being screen-printed.

In some embodiments, the edge to edge (E2E) to diameter ratio of anelectrode is about 0.5 mm to about 5 mm. In some embodiments, the E2E todiameter ratio is about 1 mm to about 4 mm. In some embodiments, the E2Eto diameter ratio is about 1 mm to about 3 mm. In some embodiments, theE2E to diameter ratio is about 1 mm to about 2 mm. In some embodiments,the E2E to diameter ratio is about 2 mm to about 5 mm. In someembodiments, the E2E to diameter ratio is about 1 mm. In someembodiments, the E2E to diameter ratio is about 2 mm. In someembodiments, the E2E to diameter ratio is about 3 mm. In someembodiments, the E2E to diameter ratio is about 4 mm. In someembodiments, the E2E to diameter ratio is about 5 mm.

In some embodiments, the electrodes disclosed herein are dry-etched. Insome embodiments, the electrodes are wet etched. In some embodiments,the electrodes undergo a combination of dry etching and wet etching.

In some embodiments, each electrode is individually site-controlled.

In some embodiments, an array of electrodes is controlled as a unit.

In some embodiments, a passivation layer is employed. In someembodiments, a passivation layer can be formed from any suitablematerial known in the art. In some embodiments, the passivation layercomprises silicon nitride. In some embodiments, the passivation layercomprises silicon dioxide. In some embodiments, the passivation layerhas a relative electrical permittivity of from about 2.0 to about 8.0.In some embodiments, the passivation layer has a relative electricalpermittivity of from about 3.0 to about 8.0, about 4.0 to about 8.0 orabout 5.0 to about 8.0. In some embodiments, the passivation layer has arelative electrical permittivity of about 2.0 to about 4.0. In someembodiments, the passivation layer has a relative electricalpermittivity of from about 2.0 to about 3.0. In some embodiments, thepassivation layer has a relative electrical permittivity of about 2.0,about 2.5, about 3.0, about 3.5 or about 4.0.

In some embodiments, the passivation layer is between about 0.1 micronsand about 10 microns in thickness. In some embodiments, the passivationlayer is between about 0.5 microns and 8 microns in thickness. In someembodiments, the passivation layer is between about 1.0 micron and 5microns in thickness. In some embodiments, the passivation layer isbetween about 1.0 micron and 4 microns in thickness. In someembodiments, the passivation layer is between about 1.0 micron and 3microns in thickness. In some embodiments, the passivation layer isbetween about 0.25 microns and 2 microns in thickness. In someembodiments, the passivation layer is between about 0.25 microns and 1micron in thickness.

In some embodiments, the passivation layer is comprised of any suitableinsulative low k dielectric material, including but not limited tosilicon nitride or silicon dioxide. In some embodiments, the passivationlayer is chosen from the group consisting of polyamids, carbon, dopedsilicon nitride, carbon doped silicon dioxide, fluorine doped siliconnitride, fluorine doped silicon dioxide, porous silicon dioxide, or anycombinations thereof. In some embodiments, the passivation layer cancomprise a dielectric ink capable of being screen-printed.

Electrode Geometry

In some embodiments, the electrodes disclosed herein can be arranged inany manner suitable for practicing the methods disclosed herein.

In some embodiments, the electrodes are in a dot configuration, e.g. theelectrodes comprises a generally circular or round configuration. Insome embodiments, the angle of orientation between dots is from about25° to about 60°. In some embodiments, the angle of orientation betweendots is from about 30° to about 55°. In some embodiments, the angle oforientation between dots is from about 30° to about 50°. In someembodiments, the angle of orientation between dots is from about 35° toabout 45°. In some embodiments, the angle of orientation between dots isabout 25°. In some embodiments, the angle of orientation between dots isabout 30°. In some embodiments, the angle of orientation between dots isabout 35°. In some embodiments, the angle of orientation between dots isabout 40°. In some embodiments, the angle of orientation between dots isabout 45°. In some embodiments, the angle of orientation between dots isabout 50°. In some embodiments, the angle of orientation between dots isabout 55°. In some embodiments, the angle of orientation between dots isabout 60°.

In some embodiments, the electrodes are in a substantially elongatedconfiguration.

In some embodiments, the electrodes are in a configuration resemblingwavy or nonlinear lines. In some embodiments, the array of electrodes isin a wavy or nonlinear line configuration, wherein the configurationcomprises a repeating unit comprising the shape of a pair of dotsconnected by a linker, wherein the dots and linker define the boundariesof the electrode, wherein the linker tapers inward towards or at themidpoint between the pair of dots, wherein the diameters of the dots arethe widest points along the length of the repeating unit, wherein theedge to edge distance between a parallel set of repeating units isequidistant, or roughly equidistant. In some embodiments, the electrodesare strips resembling wavy lines, as depicted in FIG. 8. In someembodiments, the edge to edge distance between the electrodes isequidistant, or roughly equidistant throughout the wavy lineconfiguration. In some embodiments, the use of wavy line electrodes, asdisclosed herein, lead to an enhanced DEP field gradient.

In some embodiments, the electrodes disclosed herein are in a planarconfiguration. In some embodiments, the electrodes disclosed herein arein a non-planar configuration.

In some embodiments, the devices disclosed herein surface selectivelycaptures biomolecules on its surface. For example, the devices disclosedherein may capture biomolecules, such as nucleic acids, by, for example,a. nucleic acid hybridization; b. antibody-antigen interactions; c.biotin—avidin interactions; d. ionic or electrostatic interactions; ore. any combination thereof. The devices disclosed herein, therefore, mayincorporate a functionalized surface which includes capture molecules,such as complementary nucleic acid probes, antibodies or other proteincaptures capable of capturing biomolecules (such as nucleic acids),biotin or other anchoring captures capable of capturing complementarytarget molecules such as avidin, capture molecules capable of capturingbiomolecules (such as nucleic acids) by ionic or electrostaticinteractions, or any combination thereof.

In some embodiments, the surface is functionalized to minimize and/orinhibit nonspecific binding interactions by: a. polymers (e.g.,polyethylene glycol PEG); b. ionic or electrostatic interactions; c.surfactants; or d. any combination thereof. In some embodiments, themethods disclosed herein include use of additives which reducenon-specific binding interactions by interfering in such interactions,such as Tween 20 and the like, bovine serum albumin, nonspecificimmunoglobulins, etc.

In some embodiments, the device comprises a plurality of microelectrodedevices oriented (a) flat side by side, (b) facing vertically, or (c)facing horizontally. In other embodiments, the electrodes are in asandwiched configuration, e.g. stacked on top of each other in avertical format.

Hydrogels

Overlaying electrode structures with one or more layers of materials canreduce the deleterious electrochemistry effects, including but notlimited to electrolysis reactions, heating, and chaotic fluid movementthat may occur on or near the electrodes, and still allow the effectiveseparation of cells, bacteria, virus, nanoparticles, DNA, and otherbiomolecules to be carried out. In some embodiments, the materialslayered over the electrode structures may be one or more porous layers.In other embodiments, the one or more porous layers is a polymer layer.In other embodiments, the one or more porous layers is a hydrogel.

In general, the hydrogel should have sufficient mechanical strength andbe relatively chemically inert such that it will be able to endure theelectrochemical effects at the electrode surface withoutdisconfiguration or decomposition. In general, the hydrogel issufficiently permeable to small aqueous ions, but keeps biomoleculesaway from the electrode surface.

In some embodiments, the hydrogel is a single layer, or coating.

In some embodiments, the hydrogel comprises a gradient of porosity,wherein the bottom of the hydrogel layer has greater porosity than thetop of the hydrogel layer.

In some embodiments, the hydrogel comprises multiple layers or coatings.In some embodiments, the hydrogel comprises two coats. In someembodiments, the hydrogel comprises three coats. In some embodiments,the bottom (first) coating has greater porosity than subsequentcoatings. In some embodiments, the top coat is has less porosity thanthe first coating. In some embodiments, the top coat has a mean porediameter that functions as a size cut-off for particles of greater than100 picometers in diameter.

In some embodiments, the hydrogel has a conductivity from about 0.001S/m to about 10 S/m. In some embodiments, the hydrogel has aconductivity from about 0.01 S/m to about 10 S/m. In some embodiments,the hydrogel has a conductivity from about 0.1 S/m to about 10 S/m. Insome embodiments, the hydrogel has a conductivity from about 1.0 S/m toabout 10 S/m. In some embodiments, the hydrogel has a conductivity fromabout 0.01 S/m to about 5 S/m. In some embodiments, the hydrogel has aconductivity from about 0.01 S/m to about 4 S/m. In some embodiments,the hydrogel has a conductivity from about 0.01 S/m to about 3 S/m. Insome embodiments, the hydrogel has a conductivity from about 0.01 S/m toabout 2 S/m. In some embodiments, the hydrogel has a conductivity fromabout 0.1 S/m to about 5 S/m. In some embodiments, the hydrogel has aconductivity from about 0.1 S/m to about 4 S/m. In some embodiments, thehydrogel has a conductivity from about 0.1 S/m to about 3 S/m. In someembodiments, the hydrogel has a conductivity from about 0.1 S/m to about2 S/m. In some embodiments, the hydrogel has a conductivity from about0.1 S/m to about 1.5 S/m. In some embodiments, the hydrogel has aconductivity from about 0.1 S/m to about 1.0 S/m.

In some embodiments, the hydrogel has a conductivity of about 0.1 S/m.In some embodiments, the hydrogel has a conductivity of about 0.2 S/m.In some embodiments, the hydrogel has a conductivity of about 0.3 S/m.In some embodiments, the hydrogel has a conductivity of about 0.4 S/m.In some embodiments, the hydrogel has a conductivity of about 0.5 S/m.In some embodiments, the hydrogel has a conductivity of about 0.6 S/m.In some embodiments, the hydrogel has a conductivity of about 0.7 S/m.In some embodiments, the hydrogel has a conductivity of about 0.8 S/m.In some embodiments, the hydrogel has a conductivity of about 0.9 S/m.In some embodiments, the hydrogel has a conductivity of about 1.0 S/m.

In some embodiments, the hydrogel has a thickness from about 0.1 micronsto about 10 microns. In some embodiments, the hydrogel has a thicknessfrom about 0.1 microns to about 5 microns. In some embodiments, thehydrogel has a thickness from about 0.1 microns to about 4 microns. Insome embodiments, the hydrogel has a thickness from about 0.1 microns toabout 3 microns. In some embodiments, the hydrogel has a thickness fromabout 0.1 microns to about 2 microns. In some embodiments, the hydrogelhas a thickness from about 1 micron to about 5 microns. In someembodiments, the hydrogel has a thickness from about 1 micron to about 4microns. In some embodiments, the hydrogel has a thickness from about 1micron to about 3 microns. In some embodiments, the hydrogel has athickness from about 1 micron to about 2 microns. In some embodiments,the hydrogel has a thickness from about 0.5 microns to about 1 micron.

In some embodiments, the viscosity of a hydrogel solution prior tospin-coating ranges from about 0.5 cP to about 5 cP. In someembodiments, a single coating of hydrogel solution has a viscosity ofbetween about 0.75 cP and 5 cP prior to spin-coating. In someembodiments, in a multi-coat hydrogel, the first hydrogel solution has aviscosity from about 0.5 cP to about 1.5 cP prior to spin coating. Insome embodiments, the second hydrogel solution has a viscosity fromabout 1 cP to about 3 cP. The viscosity of the hydrogel solution isbased on the polymers concentration (0.1%-10%) and polymers molecularweight (10,000 to 300,000) in the solvent and the starting viscosity ofthe solvent.

In some embodiments, the first hydrogel coating has a thickness betweenabout 0.5 microns and 1 micron. In some embodiments, the first hydrogelcoating has a thickness between about 0.5 microns and 0.75 microns. Insome embodiments, the first hydrogel coating has a thickness betweenabout 0.75 and 1 micron. In some embodiments, the second hydrogelcoating has a thickness between about 0.2 microns and 0.5 microns. Insome embodiments, the second hydrogel coating has a thickness betweenabout 0.2 and 0.4 microns. In some embodiments, the second hydrogelcoating has a thickness between about 0.2 and 0.3 microns. In someembodiments, the second hydrogel coating has a thickness between about0.3 and 0.4 microns.

In some embodiments, the hydrogel comprises any suitable syntheticpolymer forming a hydrogel. In general, any sufficiently hydrophilic andpolymerizable molecule may be utilized in the production of a syntheticpolymer hydrogel for use as disclosed herein. Polymerizable moieties inthe monomers may include alkenyl moieties including but not limited tosubstituted or unsubstituted α,β, unsaturated carbonyls wherein thedouble bond is directly attached to a carbon which is double bonded toan oxygen and single bonded to another oxygen, nitrogen, sulfur,halogen, or carbon; vinyl, wherein the double bond is singly bonded toan oxygen, nitrogen, halogen, phosphorus or sulfur; allyl, wherein thedouble bond is singly bonded to a carbon which is bonded to an oxygen,nitrogen, halogen, phosphorus or sulfur; homoallyl, wherein the doublebond is singly bonded to a carbon which is singly bonded to anothercarbon which is then singly bonded to an oxygen, nitrogen, halogen,phosphorus or sulfur; alkynyl moieties wherein a triple bond existsbetween two carbon atoms. In some embodiments, acryloyl or acrylamidomonomers such as acrylates, methacrylates, acrylamides, methacrylamides,etc., are useful for formation of hydrogels as disclosed herein. Morepreferred acrylamido monomers include acrylamides, N-substitutedacrylamides, N-substituted methacrylamides, and methacrylamide. In someembodiments, a hydrogel comprises polymers such as epoxide-basedpolymers, vinyl-based polymers, allyl-based polymers, homoallyl-basedpolymers, cyclic anhydride-based polymers, ester-based polymers,ether-based polymers, alkylene-glycol based polymers (e.g.,polypropylene glycol), and the like.

In some embodiments, the hydrogel comprises polyhydroxyethylmethacrylate(pHEMA), cellulose acetate, cellulose acetate phthalate, celluloseacetate butyrate, or any appropriate acrylamide or vinyl-based polymer,or a derivative thereof.

In some embodiments, the hydrogel is applied by vapor deposition.

In some embodiments, the hydrogel is polymerized via atom-transferradical-polymerization via (ATRP).

In some embodiments, the hydrogel is polymerized via reversibleaddition-fragmentation chain-transfer (RAFT) polymerization.

In some embodiments, additives are added to a hydrogel to increaseconductivity of the gel. In some embodiments, hydrogel additives areconductive polymers (e.g., PEDOT: PSS), salts (e.g., copper chloride),metals (e.g., gold), plasticizers (e.g., PEG200, PEG 400, or PEG 600),or co-solvents.

In some embodiments, the hydrogel also comprises compounds or materialswhich help maintain the stability of the DNA hybrids, including, but notlimited to histidine, histidine peptides, polyhistidine, lysine, lysinepeptides, and other cationic compounds or substances.

Dielectrophoretic Fields

In some embodiments, the methods, devices and systems described hereinprovide a mechanism to collect, separate, or isolate cells, particles,and/or molecules (such as nucleic acid) from a fluid material (whichoptionally contains other materials, such as contaminants, residualcellular material, or the like).

In some embodiments, an AC electrokinetic field is generated to collect,separate or isolate biomolecules, such as nucleic acids. In someembodiments, the AC electrokinetic field is a dielectrophoretic field.Accordingly, in some embodiments dielectrophoresis (DEP) is utilized invarious steps of the methods described herein.

In some embodiments, the devices and systems described herein arecapable of generating DEP fields, and the like. In specific embodiments,DEP is used to concentrate cells and/or nucleic acids (e.g.,concurrently or at different times). In certain embodiments, methodsdescribed herein further comprise energizing the array of electrodes soas to produce the first, second, and any further optional DEP fields. Insome embodiments, the devices and systems described herein are capableof being energized so as to produce the first, second, and any furtheroptional DEP fields.

DEP is a phenomenon in which a force is exerted on a dielectric particlewhen it is subjected to a non-uniform electric field. Depending on thestep of the methods described herein, aspects of the devices and systemsdescribed herein, and the like, the dielectric particle in variousembodiments herein is a biological cell and/or a molecule, such as anucleic acid molecule. Different steps of the methods described hereinor aspects of the devices or systems described herein may be utilized toisolate and separate different components, such as intact cells or otherparticular material; further, different field regions of the DEP fieldmay be used in different steps of the methods or aspects of the devicesand systems described herein. This dielectrophoretic force does notrequire the particle to be charged. In some instances, the strength ofthe force depends on the medium and the specific particles' electricalproperties, on the particles' shape and size, as well as on thefrequency of the electric field. In some instances, fields of aparticular frequency selectivity manipulate particles. In certainaspects described herein, these processes allow for the separation ofcells and/or smaller particles (such as molecules, including nucleicacid molecules) from other components (e.g., in a fluid medium) or eachother.

In various embodiments provided herein, a method described hereincomprises producing a first DEP field region and a second DEP fieldregion with the array. In various embodiments provided herein, a deviceor system described herein is capable of producing a first DEP fieldregion and a second DEP field region with the array. In some instances,the first and second field regions are part of a single field (e.g., thefirst and second regions are present at the same time, but are found atdifferent locations within the device and/or upon the array). In someembodiments, the first and second field regions are different fields(e.g. the first region is created by energizing the electrodes at afirst time, and the second region is created by energizing theelectrodes a second time). In specific aspects, the first DEP fieldregion is suitable for concentrating or isolating cells (e.g., into alow field DEP region). In some embodiments, the second DEP field regionis suitable for concentrating smaller particles, such as molecules(e.g., nucleic acid), for example into a high field DEP region. In someinstances, a method described herein optionally excludes use of eitherthe first or second DEP field region.

In some embodiments, the first DEP field region is in the same chamberof a device as disclosed herein as the second DEP field region. In someembodiments, the first DEP field region and the second DEP field regionoccupy the same area of the array of electrodes.

In some embodiments, the first DEP field region is in a separate chamberof a device as disclosed herein, or a separate device entirely, from thesecond DEP field region.

First DEP Field Region

In some aspects, e.g., high conductance buffers (>100 mS/m), the methoddescribed herein comprises applying a fluid comprising cells or otherparticulate material to a device comprising an array of electrodes, and,thereby, concentrating the cells in a first DEP field region. In someaspects, the devices and systems described herein are capable ofapplying a fluid comprising cells or other particulate material to thedevice comprising an array of electrodes, and, thereby, concentratingthe cells in a first DEP field region. Subsequent or concurrent second,or optional third and fourth DEP regions, may collect or isolate otherfluid components, including biomolecules, such as nucleic acids.

The first DEP field region may be any field region suitable forconcentrating cells from a fluid. For this application, the cells aregenerally concentrated near the array of electrodes. In someembodiments, the first DEP field region is a dielectrophoretic low fieldregion. In some embodiments, the first DEP field region is adielectrophoretic high field region. In some aspects, e.g. lowconductance buffers (<100 mS/m), the method described herein comprisesapplying a fluid comprising cells to a device comprising an array ofelectrodes, and, thereby, concentrating the cells or other particulatematerial in a first DEP field region.

In some aspects, the devices and systems described herein are capable ofapplying a fluid comprising cells or other particulate material to thedevice comprising an array of electrodes, and concentrating the cells ina first DEP field region. In various embodiments, the first DEP fieldregion may be any field region suitable for concentrating cells from afluid. In some embodiments, the cells are concentrated on the array ofelectrodes. In some embodiments, the cells are captured in adielectrophoretic high field region. In some embodiments, the cells arecaptured in a dielectrophoretic low-field region. High versus low fieldcapture is generally dependent on the conductivity of the fluid, whereingenerally, the crossover point is between about 300-500 mS/m. In someembodiments, the first DEP field region is a dielectrophoretic low fieldregion performed in fluid conductivity of greater than about 300 mS/m.In some embodiments, the first DEP field region is a dielectrophoreticlow field region performed in fluid conductivity of less than about 300mS/m. In some embodiments, the first DEP field region is adielectrophoretic high field region performed in fluid conductivity ofgreater than about 300 mS/m. In some embodiments, the first DEP fieldregion is a dielectrophoretic high field region performed in fluidconductivity of less than about 300 mS/m. In some embodiments, the firstDEP field region is a dielectrophoretic low field region performed influid conductivity of greater than about 500 mS/m. In some embodiments,the first DEP field region is a dielectrophoretic low field regionperformed in fluid conductivity of less than about 500 mS/m. In someembodiments, the first DEP field region is a dielectrophoretic highfield region performed in fluid conductivity of greater than about 500mS/m. In some embodiments, the first DEP field region is adielectrophoretic high field region performed in fluid conductivity ofless than about 500 mS/m.

In some embodiments, the first dielectrophoretic field region isproduced by an alternating current. The alternating current has anyamperage, voltage, frequency, and the like suitable for concentratingcells. In some embodiments, the first dielectrophoretic field region isproduced using an alternating current having an amperage of 0.1 microAmperes-10 Amperes; a voltage of 1-50 Volts peak to peak; and/or afrequency of 1-10,000,000 Hz. In some embodiments, the first DEP fieldregion is produced using an alternating current having a voltage of 5-25volts peak to peak. In some embodiments, the first DEP field region isproduced using an alternating current having a frequency of from 3-15kHz. In some embodiments, the first DEP field region is produced usingan alternating current having an amperage of 1 milliamp to 1 amp. Insome embodiments, the first DEP field region is produced using analternating current having an amperage of 0.1 micro Amperes-1 Ampere. Insome embodiments, the first DEP field region is produced using analternating current having an amperage of 1 micro Amperes-1 Ampere. Insome embodiments, the first DEP field region is produced using analternating current having an amperage of 100 micro Amperes-1 Ampere. Insome embodiments, the first DEP field region is produced using analternating current having an amperage of 500 micro Amperes-500 milliAmperes. In some embodiments, the first DEP field region is producedusing an alternating current having a voltage of 1-25 Volts peak topeak. In some embodiments, the first DEP field region is produced usingan alternating current having a voltage of 1-10 Volts peak to peak. Insome embodiments, the first DEP field region is produced using analternating current having a voltage of 25-50 Volts peak to peak. Insome embodiments, the first DEP field region is produced using afrequency of from 10-1,000,000 Hz. In some embodiments, the first DEPfield region is produced using a frequency of from 100-100,000 Hz. Insome embodiments, the first DEP field region is produced using afrequency of from 100-10,000 Hz. In some embodiments, the first DEPfield region is produced using a frequency of from 10,000-100,000 Hz. Insome embodiments, the first DEP field region is produced using afrequency of from 100,000-1,000,000 Hz.

In some embodiments, the first dielectrophoretic field region isproduced by a direct current. The direct current has any amperage,voltage, frequency, and the like suitable for concentrating cells. Insome embodiments, the first dielectrophoretic field region is producedusing a direct current having an amperage of 0.1 micro Amperes-1Amperes; a voltage of 10 milli Volts-10 Volts; and/or a pulse width of 1milliseconds-1000 seconds and a pulse frequency of 0.001-1000 Hz. Insome embodiments, the first DEP field region is produced using a directcurrent having an amperage of 1 micro Amperes-1 Amperes. In someembodiments, the first DEP field region is produced using a directcurrent having an amperage of 100 micro Amperes-500 milli Amperes. Insome embodiments, the first DEP field region is produced using a directcurrent having an amperage of 1 milli Amperes-1 Amperes. In someembodiments, the first DEP field region is produced using a directcurrent having an amperage of 1 micro Amperes-1 milli Amperes. In someembodiments, the first DEP field region is produced using a directcurrent having a pulse width of 500 milliseconds-500 seconds. In someembodiments, the first DEP field region is produced using a directcurrent having a pulse width of 500 milliseconds-100 seconds. In someembodiments, the first DEP field region is produced using a directcurrent having a pulse width of 1 second-1000 seconds. In someembodiments, the first DEP field region is produced using a directcurrent having a pulse width of 500 milliseconds-1 second. In someembodiments, the first DEP field region is produced using a pulsefrequency of 0.01-1000 Hz. In some embodiments, the first DEP fieldregion is produced using a pulse frequency of 0.1-100 Hz. In someembodiments, the first DEP field region is produced using a pulsefrequency of 1-100 Hz. In some embodiments, the first DEP field regionis produced using a pulse frequency of 100-1000 Hz.

In some embodiments, the fluid comprises a mixture of cell types. Forexample, blood comprises red blood cells and white blood cells.Environmental samples comprise many types of cells and other particulatematerial over a wide range of concentrations. In some embodiments, onecell type (or any number of cell types less than the total number ofcell types comprising the sample) is preferentially concentrated in thefirst DEP field. Without limitation, this embodiment is beneficial forfocusing the nucleic acid isolation procedure on a particularenvironmental contaminant, such as a fecal coliform bacterium, wherebyDNA sequencing may be used to identify the source of the contaminant. Inanother non-limiting example, the first DEP field is operated in amanner that specifically concentrates viruses and not cells (e.g., in afluid with conductivity of greater than 300 mS/m, viruses concentrate ina DEP high field region, while larger cells will concentrate in a DEPlow field region).

In some embodiments, a method, device or system described herein issuitable for isolating or separating specific cell types. In someembodiments, the DEP field of the method, device or system isspecifically tuned to allow for the separation or concentration of aspecific type of cell into a field region of the DEP field. In someembodiments, a method, device or system described herein provides morethan one field region wherein more than one type of cell is isolated orconcentrated. In some embodiments, a method, device, or system describedherein is tunable so as to allow isolation or concentration of differenttypes of cells within the DEP field regions thereof. In someembodiments, a method provided herein further comprises tuning the DEPfield. In some embodiments, a device or system provided herein iscapable of having the DEP field tuned. In some instances, such tuningmay be in providing a DEP particularly suited for the desired purpose.For example, modifications in the array, the energy, or anotherparameter are optionally utilized to tune the DEP field. Tuningparameters for finer resolution include electrode diameter, edge to edgedistance between electrodes, voltage, frequency, fluid conductivity andhydrogel composition.

In some embodiments, the first DEP field region comprises the entiretyof an array of electrodes. In some embodiments, the first DEP fieldregion comprises a portion of an array of electrodes. In someembodiments, the first DEP field region comprises about 90%, about 80%,about 70%, about 60%, about 50%, about 40%, about 30%, about 25%, about20%, or about 10% of an array of electrodes. In some embodiments, thefirst DEP field region comprises about a third of an array ofelectrodes.

Second DEP Field Region

In one aspect, following lysis of the cells (as provided below), themethods described herein involve concentrating the nucleic acid in asecond DEP field region. In another aspect, the devices and systemsdescribed herein are capable of concentrating the nucleic acid in asecond DEP field region. In some embodiments, the second DEP fieldregion is any field region suitable for concentrating nucleic acids. Insome embodiments, the nucleic acids are concentrated on the array ofelectrodes. In some embodiments, the second DEP field region is adielectrophoretic high field region. The second DEP field region is,optionally, the same as the first DEP field region.

In some embodiments, the second dielectrophoretic field region isproduced by an alternating current. In some embodiments, the alternatingcurrent has any amperage, voltage, frequency, and the like suitable forconcentrating nucleic acids. In some embodiments, the seconddielectrophoretic field region is produced using an alternating currenthaving an amperage of 0.1 micro Amperes-10 Amperes; a voltage of 1-50Volts peak to peak; and/or a frequency of 1-10,000,000 Hz. In someembodiments, the second DEP field region is produced using analternating current having an amperage of 0.1 micro Amperes-1 Ampere. Insome embodiments, the second DEP field region is produced using analternating current having an amperage of 1 micro Amperes-1 Ampere. Insome embodiments, the second DEP field region is produced using analternating current having an amperage of 100 micro Amperes-1 Ampere. Insome embodiments, the second DEP field region is produced using analternating current having an amperage of 500 micro Amperes-500 milliAmperes. In some embodiments, the second DEP field region is producedusing an alternating current having a voltage of 1-25 Volts peak topeak. In some embodiments, the second DEP field region is produced usingan alternating current having a voltage of 1-10 Volts peak to peak. Insome embodiments, the second DEP field region is produced using analternating current having a voltage of 25-50 Volts peak to peak. Insome embodiments, the second DEP field region is produced using afrequency of from 10-1,000,000 Hz. In some embodiments, the second DEPfield region is produced using a frequency of from 100-100,000 Hz. Insome embodiments, the second DEP field region is produced using afrequency of from 100-10,000 Hz. In some embodiments, the second DEPfield region is produced using a frequency of from 10,000-100,000 Hz. Insome embodiments, the second DEP field region is produced using afrequency of from 100,000-1,000,000 Hz.

In some embodiments, the second dielectrophoretic field region isproduced by a direct current. In some embodiments, the direct currenthas any amperage, voltage, frequency, and the like suitable forconcentrating nucleic acids. In some embodiments, the seconddielectrophoretic field region is produced using a direct current havingan amperage of 0.1 micro Amperes-1 Amperes; a voltage of 10 milliVolts-10 Volts; and/or a pulse width of 1 milliseconds-1000 seconds anda pulse frequency of 0.001-1000 Hz. In some embodiments, the second DEPfield region is produced using an alternating current having a voltageof 5-25 volts peak to peak. In some embodiments, the second DEP fieldregion is produced using an alternating current having a frequency offrom 3-15 kHz. In some embodiments, the second DEP field region isproduced using an alternating current having an amperage of 1 milliampto 1 amp. In some embodiments, the second DEP field region is producedusing a direct current having an amperage of 1 micro Amperes-1 Amperes.In some embodiments, the second DEP field region is produced using adirect current having an amperage of 100 micro Amperes-500 milliAmperes. In some embodiments, the second DEP field region is producedusing a direct current having an amperage of 1 milli Amperes-1 Amperes.In some embodiments, the second DEP field region is produced using adirect current having an amperage of 1 micro Amperes-1 milli Amperes. Insome embodiments, the second DEP field region is produced using a directcurrent having a pulse width of 500 milliseconds-500 seconds. In someembodiments, the second DEP field region is produced using a directcurrent having a pulse width of 500 milliseconds-100 seconds. In someembodiments, the second DEP field region is produced using a directcurrent having a pulse width of 1 second-1000 seconds. In someembodiments, the second DEP field region is produced using a directcurrent having a pulse width of 500 milliseconds-1 second. In someembodiments, the second DEP field region is produced using a pulsefrequency of 0.01-1000 Hz. In some embodiments, the second DEP fieldregion is produced using a pulse frequency of 0.1-100 Hz. In someembodiments, the second DEP field region is produced using a pulsefrequency of 1-100 Hz. In some embodiments, the second DEP field regionis produced using a pulse frequency of 100-1000 Hz.

In some embodiments, the second DEP field region comprises the entiretyof an array of electrodes. In some embodiments, the second DEP fieldregion comprises a portion of an array of electrodes. In someembodiments, the second DEP field region comprises about 90%, about 80%,about 70%, about 60%, about 50%, about 40%, about 30%, about 25%, about20%, or about 10% of an array of electrodes. In some embodiments, thesecond DEP field region comprises about a third of an array ofelectrodes.

Isolating Nucleic Acids

In one aspect, described herein is a method for isolating a nucleic acidfrom a fluid comprising cells. In some embodiments, the nucleic acidsare initially inside the cells. As seen in FIG. 5, the method comprisesconcentrating the cells near a high field region in some instances. Insome embodiments, disclosed herein is method for isolating a nucleicacid from a fluid comprising cells, the method comprising: a. applyingthe fluid to a device, the device comprising an array of electrodes; b.concentrating a plurality of cells in a first AC electrokinetic (e.g.,dielectrophoretic) field region; c. isolating nucleic acid in a secondAC electrokinetic (e.g., dielectrophoretic) field region; and d.flushing cells away. In some instances, the cells are lysed in the highfield region. Following lysis, the nucleic acids remain in the highfield region and/or are concentrated in the high field region. In someinstances, residual cellular material is concentrated near the low fieldregion. In some embodiments, the residual material is washed from thedevice and/or washed from the nucleic acids. In some embodiments, thenucleic acid is concentrated in the second AC electrokinetic fieldregion.

In one aspect, described herein is a method for isolating a nucleic acidfrom a fluid comprising cells or other particulate material. In someembodiments, the nucleic acids are not inside the cells (e.g., cell-freeDNA in fluid). In some embodiments, disclosed herein is a method forisolating a nucleic acid from a fluid comprising cells or otherparticulate material, the method comprising: a. applying the fluid to adevice, the device comprising an array of electrodes; b. concentrating aplurality of cells in a first AC electrokinetic (e.g.,dielectrophoretic) field region; c. isolating nucleic acid in a secondAC electrokinetic (e.g., dielectrophoretic) field region; and d.flushing cells away. In some embodiments, the method further comprisesdegrading residual proteins after flushing cells away. FIG. 6 shows anexemplary method for isolating extra-cellular nucleic acids from a fluidcomprising cells. In some embodiments, cells are concentrated on or neara low field region and nucleic acids are concentrated on or near a highfield region. In some instances, the cells are washed from the deviceand/or washed from the nucleic acids.

In one aspect, the methods, systems and devices described herein isolatenucleic acid from a fluid comprising cells or other particulatematerial. In one aspect, dielectrophoresis is used to concentrate cells.In some embodiments, the fluid is a liquid, optionally water or anaqueous solution or dispersion. In some embodiments, the fluid is anysuitable fluid including a bodily fluid. Exemplary bodily fluids includewhole blood, serum, plasma, bile, milk, cerebrospinal fluid, gastricjuice, ejaculate, mucus, peritoneal fluid, saliva, sweat, tears, urine,and other bodily fluids. In some embodiments, nucleic acids are isolatedfrom bodily fluids using the methods, systems or devices describedherein as part of a medical therapeutic or diagnostic procedure, deviceor system. In some embodiments, the fluid is tissues and/or cellssolubilized and/or dispersed in a fluid. For example, the tissue can bea cancerous tumor from which nucleic acid can be isolated using themethods, devices or systems described herein.

In some embodiments, the fluid is an environmental sample. In someembodiments, the environmental sample is assayed or monitored for thepresence of a particular nucleic acid sequence indicative of a certaincontamination, infestation incidence or the like. The environmentalsample can also be used to determine the source of a certaincontamination, infestation incidence or the like using the methods,devices or systems described herein. Exemplary environmental samplesinclude municipal wastewater, industrial wastewater, water or fluid usedin or produced as a result of various manufacturing processes, lakes,rivers, oceans, aquifers, ground water, storm water, plants or portionsof plants, animals or portions of animals, insects, municipal watersupplies, and the like.

In some embodiments, the fluid is a food or beverage. The food orbeverage can be assayed or monitored for the presence of a particularnucleic acid sequence indicative of a certain contamination, infestationincidence or the like. The food or beverage can also be used todetermine the source of a certain contamination, infestation incidenceor the like using the methods, devices or systems described herein. Invarious embodiments, the methods, devices and systems described hereincan be used with one or more of bodily fluids, environmental samples,and foods and beverages to monitor public health or respond to adversepublic health incidences.

In some embodiments, the fluid is a growth medium. The growth medium canbe any medium suitable for culturing cells, for example lysogeny broth(LB) for culturing E. coli, Ham's tissue culture medium for culturingmammalian cells, and the like. The medium can be a rich medium, minimalmedium, selective medium, and the like. In some embodiments, the mediumcomprises or consists essentially of a plurality of clonal cells. Insome embodiments, the medium comprises a mixture of at least twospecies.

In some embodiments, the fluid is water.

The cells are any cell suitable for isolating nucleic acids from asdescribed herein. In various embodiments, the cells are eukaryotic orprokaryotic. In various embodiments, the cells consist essentially of aplurality of clonal cells or may comprise at least two species and/or atleast two strains.

In various embodiments, the cells are pathogen cells, bacteria cells,plant cells, animal cells, insect cells, algae cells, cyanobacterialcells, organelles and/or combinations thereof. As used herein, “cells”include viruses and other intact pathogenic microorganisms. The cellscan be microorganisms or cells from multi-cellular organisms. In someinstances, the cells originate from a solubilized tissue sample.

In various embodiments, the cells are wild-type or geneticallyengineered. In some instances, the cells comprise a library of mutantcells. In some embodiments, the cells are randomly mutagenized such ashaving undergone chemical mutagenesis, radiation mutagenesis (e.g. UVradiation), or a combination thereof. In some embodiments, the cellshave been transformed with a library of mutant nucleic acid molecules.

In some embodiments, the fluid may also comprise other particulatematerial. Such particulate material may be, for example, inclusionbodies (e.g., ceroids or Mallory bodies), cellular casts (e.g., granularcasts, hyaline casts, cellular casts, waxy casts and pseudo casts),Pick's bodies, Lewy bodies, fibrillary tangles, fibril formations,cellular debris and other particulate material. In some embodiments,particulate material is an aggregated protein (e.g., beta-amyloid).

The fluid can have any conductivity including a high or lowconductivity. In some embodiments, the conductivity is between about 1μS/m to about 10 mS/m. In some embodiments, the conductivity is betweenabout 10 μS/m to about 10 mS/m. In other embodiments, the conductivityis between about 50 μS/m to about 10 mS/m. In yet other embodiments, theconductivity is between about 100 μS/m to about 10 mS/m, between about100 μS/m to about 8 mS/m, between about 100 μS/m to about 6 mS/m,between about 100 μS/m to about 5 mS/m, between about 100 μS/m to about4 mS/m, between about 100 μS/m to about 3 mS/m, between about 100 μS/mto about 2 mS/m, or between about 100 μS/m to about 1 mS/m.

In some embodiments, the conductivity is about 1 μS/m. In someembodiments, the conductivity is about 10 μS/m. In some embodiments, theconductivity is about 100 μS/m. In some embodiments, the conductivity isabout 1 mS/m. In other embodiments, the conductivity is about 2 mS/m. Insome embodiments, the conductivity is about 3 mS/m. In yet otherembodiments, the conductivity is about 4 mS/m. In some embodiments, theconductivity is about 5 mS/m. In some embodiments, the conductivity isabout 10 mS/m. In still other embodiments, the conductivity is about 100mS/m. In some embodiments, the conductivity is about 1 S/m. In otherembodiments, the conductivity is about 10 S/m.

In some embodiments, the conductivity is at least 1 μS/m. In yet otherembodiments, the conductivity is at least 10 μS/m. In some embodiments,the conductivity is at least 100 μS/m. In some embodiments, theconductivity is at least 1 mS/m. In additional embodiments, theconductivity is at least 10 mS/m. In yet other embodiments, theconductivity is at least 100 mS/m. In some embodiments, the conductivityis at least 1 S/m. In some embodiments, the conductivity is at least 10S/m. In some embodiments, the conductivity is at most 1 μS/m. In someembodiments, the conductivity is at most 10 μS/m. In other embodiments,the conductivity is at most 100 μS/m. In some embodiments, theconductivity is at most 1 mS/m. In some embodiments, the conductivity isat most 10 mS/m. In some embodiments, the conductivity is at most 100mS/m. In yet other embodiments, the conductivity is at most 1 S/m. Insome embodiments, the conductivity is at most 10 S/m.

In some embodiments, the fluid is a small volume of liquid includingless than 10 ml. In some embodiments, the fluid is less than 8 ml. Insome embodiments, the fluid is less than 5 ml. In some embodiments, thefluid is less than 2 ml. In some embodiments, the fluid is less than 1ml. In some embodiments, the fluid is less than 500 μl. In someembodiments, the fluid is less than 200 μl. In some embodiments, thefluid is less than 100 μl. In some embodiments, the fluid is less than50 μl. In some embodiments, the fluid is less than 10 μl. In someembodiments, the fluid is less than 5 μl. In some embodiments, the fluidis less than 1 μl. In preferred embodiments, the fluid is between about50 μl to about 500 μl.

In some embodiments, the quantity of fluid applied to the device or usedin the method comprises less than about 100,000,000 cells. In someembodiments, the fluid comprises less than about 10,000,000 cells. Insome embodiments, the fluid comprises less than about 1,000,000 cells.In some embodiments, the fluid comprises less than about 100,000 cells.In some embodiments, the fluid comprises less than about 10,000 cells.In some embodiments, the fluid comprises less than about 1,000 cells.

In some embodiments, isolation of nucleic acid from a fluid comprisingcells or other particulate material with the devices, systems andmethods described herein takes less than about 30 minutes, less thanabout 20 minutes, less than about 15 minutes, less than about 10minutes, less than about 5 minutes or less than about 1 minute. In otherembodiments, isolation of nucleic acid from a fluid comprising cells orother particulate material with the devices, systems and methodsdescribed herein takes not more than 30 minutes, not more than about 20minutes, not more than about 15 minutes, not more than about 10 minutes,not more than about 5 minutes, not more than about 2 minutes or not morethan about 1 minute. In additional embodiments, isolation of nucleicacid from a fluid comprising cells or other particulate material withthe devices, systems and methods described herein takes less than about15 minutes, preferably less than about 10 minutes or less than about 5minutes.

In some instances, extra-cellular DNA or other nucleic acid (outsidecells) is isolated from a fluid comprising cells of other particulatematerial. In some embodiments, the fluid comprises cells. In someembodiments, the fluid does not comprise cells.

Cell Lysis

In one aspect, following concentrating the cells in a firstdielectrophoretic field region, the method involves freeing nucleicacids from the cells. In another aspect, the devices and systemsdescribed herein are capable of freeing nucleic acids from the cells. Insome embodiments, the nucleic acids are freed from the cells in thefirst DEP field region.

In some embodiments, the methods described herein free nucleic acidsfrom a plurality of cells by lysing the cells. In some embodiments, thedevices and systems described herein are capable of freeing nucleicacids from a plurality of cells by lysing the cells. One method of celllysis involves applying a direct current to the cells after isolation ofthe cells on the array. The direct current has any suitable amperage,voltage, and the like suitable for lysing cells. In some embodiments,the current has a voltage of about 1 Volt to about 500 Volts. In someembodiments, the current has a voltage of about 10 Volts to about 500Volts. In other embodiments, the current has a voltage of about 10 Voltsto about 250 Volts. In still other embodiments, the current has avoltage of about 50 Volts to about 150 Volts. Voltage is generally thedriver of cell lysis, as high electric fields result in failed membraneintegrity.

In some embodiments, the direct current used for lysis comprises one ormore pulses having any duration, frequency, and the like suitable forlysing cells. In some embodiments, a voltage of about 100 volts isapplied for about 1 millisecond to lyse cells. In some embodiments, thevoltage of about 100 volts is applied 2 or 3 times over the source of asecond.

In some embodiments, the frequency of the direct current depends onvolts/cm, pulse width, and the fluid conductivity. In some embodiments,the pulse has a frequency of about 0.001 to about 1000 Hz. In someembodiments, the pulse has a frequency from about 10 to about 200 Hz. Inother embodiments, the pulse has a frequency of about 0.01 Hz-1000 Hz.In still other embodiments, the pulse has a frequency of about 0.1Hz-1000 Hz, about 1 Hz-1000 Hz, about 1 Hz-500 Hz, about 1 Hz-400 Hz,about 1 Hz-300 Hz, or about 1 Hz-about 250 Hz. In some embodiments, thepulse has a frequency of about 0.1 Hz. In other embodiments, the pulsehas a frequency of about 1 Hz. In still other embodiments, the pulse hasa frequency of about 5 Hz, about 10 Hz, about 50 Hz, about 100 Hz, about200 Hz, about 300 Hz, about 400 Hz, about 500 Hz, about 600 Hz, about700 Hz, about 800 Hz, about 900 Hz or about 1000 Hz.

In other embodiments, the pulse has a duration of about 1 millisecond(ms)-1000 seconds (s). In some embodiments, the pulse has a duration ofabout 10 ms-1000 s. In still other embodiments, the pulse has a durationof about 100 ms-1000 s, about 1 s-1000 s, about 1 s-500 s, about 1 s-250s or about 1 s-150 s. In some embodiments, the pulse has a duration ofabout 1 ms, about 10 ms, about 100 ms, about 1 s, about 2 s, about 3 s,about 4 s, about 5 s, about 6 s, about 7 s, about 8 s, about 9 s, about10 s, about 20 s, about 50 s, about 100 s, about 200 s, about 300 s,about 500 s or about 1000 s. In some embodiments, the pulse has afrequency of 0.2 to 200 Hz with duty cycles from 10-50%.

In some embodiments, the direct current is applied once, or as multiplepulses. Any suitable number of pulses may be applied including about1-20 pulses. There is any suitable amount of time between pulsesincluding about 1 millisecond-1000 seconds. In some embodiments, thepulse duration is 0.01 to 10 seconds.

In some embodiments, the cells are lysed using other methods incombination with a direct current applied to the isolated cells. In yetother embodiments, the cells are lysed without use of direct current. Invarious aspects, the devices and systems are capable of lysing cellswith direct current in combination with other means, or may be capableof lysing cells without the use of direct current. Any method of celllysis known to those skilled in the art may be suitable including, butnot limited to application of a chemical lysing agent (e.g., an acid),an enzymatic lysing agent, heat, pressure, shear force, sonic energy,osmotic shock, or combinations thereof. Lysozyme is an example of anenzymatic-lysing agent.

Removal of Residual Material

In some embodiments, following concentration of the nucleic acids in thesecond DEP field region, the method includes optionally flushingresidual material from the nucleic acid. In some embodiments, thedevices or systems described herein are capable of optionally and/orcomprising a reservoir comprising a fluid suitable for flushing residualmaterial from the nucleic acid. In some embodiments, the nucleic acid isheld near the array of electrodes, such as in the second DEP fieldregion, by continuing to energize the electrodes. “Residual material” isanything originally present in the fluid, originally present in thecells, added during the procedure, created through any step of theprocess including but not limited to lysis of the cells (i.e. residualcellular material), and the like. For example, residual materialincludes non-lysed cells, cell wall fragments, proteins, lipids,carbohydrates, minerals, salts, buffers, plasma, and undesired nucleicacids. In some embodiments, the lysed cellular material comprisesresidual protein freed from the plurality of cells upon lysis. It ispossible that not all of the nucleic acid will be concentrated in thesecond DEP field. In some embodiments, a certain amount of nucleic acidis flushed with the residual material.

In some embodiments, the residual material is flushed in any suitablefluid, for example in water, TBE buffer, or the like. In someembodiments, the residual material is flushed with any suitable volumeof fluid, flushed for any suitable period of time, flushed with morethan one fluid, or any other variation. In some embodiments, the methodof flushing residual material is related to the desired level ofisolation of the nucleic acid, with higher purity nucleic acid requiringmore stringent flushing and/or washing. In other embodiments, the methodof flushing residual material is related to the particular startingmaterial and its composition. In some instances, a starting materialthat is high in lipid requires a flushing procedure that involves ahydrophobic fluid suitable for solubilizing lipids.

In some embodiments, the method includes degrading residual materialincluding residual protein. In some embodiments, the devices or systemsare capable of degrading residual material including residual protein.For example, proteins are degraded by one or more of chemicaldegradation (e.g. acid hydrolysis) and enzymatic degradation. In someembodiments, the enzymatic degradation agent is a protease. In otherembodiments, the protein degradation agent is Proteinase K. The optionalstep of degradation of residual material is performed for any suitabletime, temperature, and the like. In some embodiments, the degradedresidual material (including degraded proteins) is flushed from thenucleic acid.

In some embodiments, the agent used to degrade the residual material isinactivated or degraded. In some embodiments, the devices or systems arecapable of degrading or inactivating the agent used to degrade theresidual material. In some embodiments, an enzyme used to degrade theresidual material is inactivated by heat (e.g., 50 to 95° C. for 5-15minutes). For example, enzymes including proteases, (for example,Proteinase K) are degraded and/or inactivated using heat (typically, 15minutes, 70° C.). In some embodiments wherein the residual proteins aredegraded by an enzyme, the method further comprises inactivating thedegrading enzyme (e.g., Proteinase K) following degradation of theproteins. In some embodiments, heat is provided by a heating module inthe device (temperature range, e.g., from 30 to 95° C.).

The order and/or combination of certain steps of the method can bevaried. In some embodiments, the devices or methods are capable ofperforming certain steps in any order or combination. For example, insome embodiments, the residual material and the degraded proteins areflushed in separate or concurrent steps. That is, the residual materialis flushed, followed by degradation of residual proteins, followed byflushing degraded proteins from the nucleic acid. In some embodiments,one first degrades the residual proteins, and then flush both theresidual material and degraded proteins from the nucleic acid in acombined step.

In some embodiments, the nucleic acid are retained in the device andoptionally used in further procedures such as PCR or other proceduresmanipulating or amplifying nucleic acid. In some embodiments, thedevices and systems are capable of performing PCR or other optionalprocedures. In other embodiments, the nucleic acids are collected and/oreluted from the device. In some embodiments, the devices and systems arecapable of allowing collection and/or elution of nucleic acid from thedevice or system. In some embodiments, the isolated nucleic acid iscollected by (i) turning off the second dielectrophoretic field region;and (ii) eluting the nucleic acid from the array in an eluant. Exemplaryeluants include water, TE, TBE and L-Histidine buffer.

Nucleic Acids and Yields Thereof

In some embodiments, the method, device, or system described herein isoptionally utilized to obtain, isolate, or separate any desired nucleicacid that may be obtained from such a method, device or system. Nucleicacids isolated by the methods, devices and systems described hereininclude DNA (deoxyribonucleic acid), RNA (ribonucleic acid), andcombinations thereof. In some embodiments, the nucleic acid is isolatedin a form suitable for sequencing or further manipulation of the nucleicacid, including amplification, ligation or cloning.

In various embodiments, an isolated or separated nucleic acid is acomposition comprising nucleic acid that is free from at least 99% bymass of other materials, free from at least 99% by mass of residualcellular material (e.g., from lysed cells from which the nucleic acid isobtained), free from at least 98% by mass of other materials, free fromat least 98% by mass of residual cellular material, free from at least95% by mass of other materials, free from at least 95% by mass ofresidual cellular material, free from at least 90% by mass of othermaterials, free from at least 90% by mass of residual cellular material,free from at least 80% by mass of other materials, free from at least80% by mass of residual cellular material, free from at least 70% bymass of other materials, free from at least 70% by mass of residualcellular material, free from at least 60% by mass of other materials,free from at least 60% by mass of residual cellular material, free fromat least 50% by mass of other materials, free from at least 50% by massof residual cellular material, free from at least 30% by mass of othermaterials, free from at least 30% by mass of residual cellular material,free from at least 10% by mass of other materials, free from at least10% by mass of residual cellular material, free from at least 5% by massof other materials, or free from at least 5% by mass of residualcellular material.

In various embodiments, the nucleic acid has any suitable purity. Forexample, if a DNA sequencing procedure can work with nucleic acidsamples having about 20% residual cellular material, then isolation ofthe nucleic acid to 80% is suitable. In some embodiments, the isolatednucleic acid comprises less than about 80%, less than about 70%, lessthan about 60%, less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, less than about 10%, less than about 5%,or less than about 2% non-nucleic acid cellular material and/or proteinby mass. In some embodiments, the isolated nucleic acid comprisesgreater than about 99%, greater than about 98%, greater than about 95%,greater than about 90%, greater than about 80%, greater than about 70%,greater than about 60%, greater than about 50%, greater than about 40%,greater than about 30%, greater than about 20%, or greater than about10% nucleic acid by mass.

The nucleic acids are isolated in any suitable form includingunmodified, derivatized, fragmented, non-fragmented, and the like. Insome embodiments, the nucleic acid is collected in a form suitable forsequencing. In some embodiments, the nucleic acid is collected in afragmented form suitable for shotgun-sequencing, amplification or othermanipulation. The nucleic acid may be collected from the device in asolution comprising reagents used in, for example, a DNA sequencingprocedure, such as nucleotides as used in sequencing by synthesismethods.

In some embodiments, the methods described herein result in an isolatednucleic acid sample that is approximately representative of the nucleicacid of the starting sample. In some embodiments, the devices andsystems described herein are capable of isolating nucleic acid from asample that is approximately representative of the nucleic acid of thestarting sample. That is, the population of nucleic acids collected bythe method, or capable of being collected by the device or system, aresubstantially in proportion to the population of nucleic acids presentin the cells in the fluid. In some embodiments, this aspect isadvantageous in applications in which the fluid is a complex mixture ofmany cell types and the practitioner desires a nucleic acid-basedprocedure for determining the relative populations of the various celltypes.

In some embodiments, the nucleic acid isolated using the methodsdescribed herein or capable of being isolated by the devices describedherein is high-quality and/or suitable for using directly in downstreamprocedures such as DNA sequencing, nucleic acid amplification, such asPCR, or other nucleic acid manipulation, such as ligation, cloning orfurther translation or transformation assays. In some embodiments, thecollected nucleic acid comprises at most 0.01% protein. In someembodiments, the collected nucleic acid comprises at most 0.5% protein.In some embodiments, the collected nucleic acid comprises at most 0.1%protein. In some embodiments, the collected nucleic acid comprises atmost 1% protein. In some embodiments, the collected nucleic acidcomprises at most 2% protein. In some embodiments, the collected nucleicacid comprises at most 3% protein. In some embodiments, the collectednucleic acid comprises at most 4% protein. In some embodiments, thecollected nucleic acid comprises at most 5% protein.

In some embodiments, the nucleic acid isolated by the methods describedherein or capable of being isolated by the devices described herein hasa concentration of at least 0.5 ng/mL. In some embodiments, the nucleicacid isolated by the methods described herein or capable of beingisolated by the devices described herein has a concentration of at least1 ng/mL. In some embodiments, the nucleic acid isolated by the methodsdescribed herein or capable of being isolated by the devices describedherein has a concentration of at least 5 ng/mL. In some embodiments, thenucleic acid isolated by the methods described herein or capable ofbeing isolated by the devices described herein has a concentration of atleast 10 ng/ml.

In some embodiments, about 50 pico-grams of nucleic acid is isolatedfrom about 5,000 cells using the methods, systems or devices describedherein. In some embodiments, the methods, systems or devices describedherein yield at least 10 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 20 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 50 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 75 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 100 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 200 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 300 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 400 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 500 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 1,000 pico-grams of nucleic acid from about 5,000cells. In some embodiments, the methods, systems or devices describedherein yield at least 10,000 pico-grams of nucleic acid from about 5,000cells.

Assays and Applications

In some embodiments, the methods described herein further compriseoptionally amplifying the isolated nucleic acid by polymerase chainreaction (PCR). In some embodiments, the PCR reaction is performed on ornear the array of electrodes or in the device. In some embodiments, thedevice or system comprise a heater and/or temperature control mechanismssuitable for thermocycling.

PCR is optionally done using traditional thermocycling by placing thereaction chemistry analytes in between two efficient thermoconductiveelements (e.g., aluminum or silver) and regulating the reactiontemperatures using TECs. Additional designs optionally use infraredheating through optically transparent material like glass or thermopolymers. In some instances, designs use smart polymers or smart glassthat comprise conductive wiring networked through the substrate. Thisconductive wiring enables rapid thermal conductivity of the materialsand (by applying appropriate DC voltage) provides the requiredtemperature changes and gradients to sustain efficient PCR reactions. Incertain instances, heating is applied using resistive chip heaters andother resistive elements that will change temperature rapidly andproportionally to the amount of current passing through them.

In some embodiments, used in conjunction with traditional fluorometry(ccd, pmt, other optical detector, and optical filters), foldamplification is monitored in real-time or on a timed interval. Incertain instances, quantification of final fold amplification isreported via optical detection converted to AFU (arbitrary fluorescenceunits correlated to analyze doubling) or translated to electrical signalvia impedance measurement or other electrochemical sensing.

Given the small size of the micro electrode array, these elements areoptionally added around the micro electrode array and the PCR reactionwill be performed in the main sample processing chamber (over the DEParray) or the analytes to be amplified are optionally transported viafluidics to another chamber within the fluidic cartridge to enableon-cartridge Lab-On-Chip processing.

In some instances, light delivery schemes are utilized to provide theoptical excitation and/or emission and/or detection of foldamplification. In certain embodiments, this includes using the flow cellmaterials (thermal polymers like acrylic (PMMA) cyclic olefin polymer(COP), cyclic olefin co-polymer, (COC), etc.) as optical wave guides toremove the need to use external components. In addition, in someinstances light sources—light emitting diodes—LEDs, vertical-cavitysurface-emitting lasers—VCSELs, and other lighting schemes areintegrated directly inside the flow cell or built directly onto themicro electrode array surface to have internally controlled and poweredlight sources. Miniature PMTs, CCDs, or CMOS detectors can also be builtinto the flow cell. This minimization and miniaturization enablescompact devices capable of rapid signal delivery and detection whilereducing the footprint of similar traditional devices (i.e. a standardbench top PCR/QPCR/Fluorometer).

Amplification on Chip

In some instances, silicon microelectrode arrays can withstand thermalcycling necessary for PCR. In some applications, on-chip PCR isadvantageous because small amounts of target nucleic acids can be lostduring transfer steps. In certain embodiments of devices, systems orprocesses described herein, any one or more of multiple PCR techniquesare optionally used, such techniques optionally including any one ormore of the following: thermal cycling in the flowcell directly; movingthe material through microchannels with different temperature zones; andmoving volume into a PCR tube that can be amplified on system ortransferred to a PCR machine. In some instances, droplet PCR isperformed if the outlet contains a T-junction that contains animmiscible fluid and interfacial stabilizers (surfactants, etc). Incertain embodiments, droplets are thermal cycled in by any suitablemethod.

In some embodiments, amplification is performed using an isothermalreaction, for example, transcription mediated amplification, nucleicacid sequence-based amplification, signal mediated amplification of RNAtechnology, strand displacement amplification, rolling circleamplification, loop-mediated isothermal amplification of DNA, isothermalmultiple displacement amplification, helicase-dependent amplification,single primer isothermal amplification or circular helicase-dependentamplification.

In various embodiments, amplification is performed in homogenoussolution or as heterogeneous system with anchored primer(s). In someembodiments of the latter, the resulting amplicons are directly linkedto the surface for higher degree of multiplex. In some embodiments, theamplicon is denatured to render single stranded products on or near theelectrodes. Hybridization reactions are then optionally performed tointerrogate the genetic information, such as single nucleotidepolymorphisms (SNPs), Short Tandem Repeats (STRs), mutations,insertions/deletions, methylation, etc. Methylation is optionallydetermined by parallel analysis where one DNA sample is bisulfitetreated and one is not. Bisulfite depurinates unmodified C becoming a U.Methylated C is unaffected in some instances. In some embodiments,allele specific base extension is used to report the base of interest.

Rather than specific interactions, the surface is optionally modifiedwith nonspecific moieties for capture. For example, surface could bemodified with polycations, i.e., polylysine, to capture DNA moleculeswhich can be released by reverse bias (−V). In some embodiments,modifications to the surface are uniform over the surface or patternedspecifically for functionalizing the electrodes or non electroderegions. In certain embodiments, this is accomplished withphotolithography, electrochemical activation, spotting, and the like.

In some applications, where multiple chip designs are employed, it isadvantageous to have a chip sandwich where the two devices are facingeach other, separated by a spacer, to form the flow cell. In variousembodiments, devices are run sequentially or in parallel. For sequencingand next generation sequencing (NGS), size fragmentation and selectionhas ramifications on sequencing efficiency and quality. In someembodiments, multiple chip designs are used to narrow the size range ofmaterial collected creating a band pass filter. In some instances,current chip geometry (e.g., 80 um diameter electrodes on 200 umcenter-center pitch (80/200) acts as 500 bp cutoff filter (e.g., usingvoltage and frequency conditions around 10 Vpp and 10 kHz). In suchinstances, a nucleic acid of greater than 500 bp is captured, and anucleic acid of less than 500 bp is not. Alternate electrode diameterand pitch geometries have different cutoff sizes such that a combinationof chips should provide a desired fragment size. In some instances, a 40um diameter electrode on 100 um center-center pitch (40/100) has a lowercutoff threshold, whereas a 160 um diameter electrode on 400 umcenter-center pitch (160/400) has a higher cutoff threshold relative tothe 80/200 geometry, under similar conditions. In various embodiments,geometries on a single chip or multiple chips are combined to select fora specific sized fragments or particles. For example a 600 bp cutoffchip would leave a nucleic acid of less than 600 bp in solution, thenthat material is optionally recaptured with a 500 bp cutoff chip (whichis opposing the 600 bp chip). This leaves a nucleic acid populationcomprising 500-600 bp in solution. This population is then optionallyamplified in the same chamber, a side chamber, or any otherconfiguration. In some embodiments, size selection is accomplished usinga single electrode geometry, wherein nucleic acid of >500 bp is isolatedon the electrodes, followed by washing, followed by reduction of the ACEhigh field strength (change voltage, frequency, conductivity) in orderto release nucleic acids of <600 bp, resulting in a supernatant nucleicacid population between 500-600 bp.

In some embodiments, the chip device is oriented vertically with aheater at the bottom edge which creates a temperature gradient column.In certain instances, the bottom is at denaturing temperature, themiddle at annealing temperature, the top at extension temperature. Insome instances, convection continually drives the process. In someembodiments, provided herein are methods or systems comprising anelectrode design that specifically provides for electrothermal flows andacceleration of the process. In some embodiments, such design isoptionally on the same device or on a separate device positionedappropriately. In some instances, active or passive cooling at the top,via fins or fans, or the like. provides a steep temperature gradient. Insome instances the device or system described herein comprises, or amethod described herein uses, temperature sensors on the device or inthe reaction chamber monitor temperature and such sensors are optionallyused to adjust temperature on a feedback basis. In some instances, suchsensors are coupled with materials possessing different thermal transferproperties to create continuous and/or discontinuous gradient profiles.

In some embodiments, the amplification proceeds at a constanttemperature (i.e, isothermal amplification).

In some embodiments, the methods disclosed herein further comprisesequencing the nucleic acid isolated as disclosed herein. In someembodiments, the nucleic acid is sequenced by Sanger sequencing or nextgeneration sequencing (NGS). In some embodiments, the next generationsequencing methods include, but are not limited to, pyrosequencing, ionsemiconductor sequencing, polony sequencing, sequencing by ligation, DNAnanoball sequencing, sequencing by ligation, or single moleculesequencing.

In some embodiments, the isolated nucleic acids disclosed herein areused in Sanger sequencing. In some embodiments, Sanger sequencing isperformed within the same device as the nucleic acid isolation(Lab-on-Chip). Lab-on-Chip workflow for sample prep and Sangersequencing results would incorporate the following steps: a) sampleextraction using ACE chips; b) performing amplification of targetsequences on chip; c) capture PCR products by ACE; d) perform cyclesequencing to enrich target strand; e) capture enriched target strands;f) perform Sanger chain termination reactions; perform electrophoreticseparation of target sequences by capillary electrophoresis with on chipmulti-color fluorescence detection. Washing nucleic acids, addingreagent, and turning off voltage is performed as necessary. Reactionscan be performed on a single chip with plurality of capture zones or onseparate chips and/or reaction chambers.

In some embodiments, the method disclosed herein further compriseperforming a reaction on the nucleic acids (e.g., fragmentation,restriction digestion, ligation of DNA or RNA). In some embodiments, thereaction occurs on or near the array or in a device, as disclosedherein.

Other Assays

The isolated nucleic acids disclosed herein may be further utilized in avariety of assay formats. For instance, devices which are addressed withnucleic acid probes or amplicons may be utilized in dot blot or reversedot blot analyses, base-stacking single nucleotide polymorphism (SNP)analysis, SNP analysis with electronic stringency, or in STR analysis.In addition, such devices disclosed herein may be utilized in formatsfor enzymatic nucleic acid modification, or protein-nucleic acidinteraction, such as, e.g., gene expression analysis with enzymaticreporting, anchored nucleic acid amplification, or other nucleic acidmodifications suitable for solid-phase formats including restrictionendonuclease cleavage, endo- or exo-nuclease cleavage, minor groovebinding protein assays, terminal transferase reactions, polynucleotidekinase or phosphatase reactions, ligase reactions, topoisomerasereactions, and other nucleic acid binding or modifying proteinreactions.

In addition, the devices disclosed herein can be useful in immunoassays.For instance, in some embodiments, locations of the devices can belinked with antigens (e.g., peptides, proteins, carbohydrates, lipids,proteoglycans, glycoproteins, etc.) in order to assay for antibodies ina bodily fluid sample by sandwich assay, competitive assay, or otherformats. Alternatively, the locations of the device may be addressedwith antibodies, in order to detect antigens in a sample by sandwichassay, competitive assay, or other assay formats. As the isoelectricpoint of antibodies and proteins can be determined fairly easily byexperimentation or pH/charge computations, the electronic addressing andelectronic concentration advantages of the devices may be utilized bysimply adjusting the pH of the buffer so that the addressed or analytespecies will be charged.

In some embodiments, the isolated nucleic acids are useful for use inimmunoassay-type arrays or nucleic acid arrays.

Exemplary Comparison

Approximately 100 ng of input E. coli genome is necessary forconventional manual methods, (e.g., 50 ng of input DNA is required forNextera, assuming 50% recovery (Epicentre WaterMaster kit claimsrecovery about 30-60%) from DNA extraction purification). This isequivalent to about 20 million bacteria. In some embodiments of thepresent invention, less than 10,000 bacteria input is sufficient (e.g.,since the chip is self contained and involves less transfers theefficiency is higher). In some embodiments, this is at least a 100 foldreduction in input, which can be important for applications where sampleis limited, such as tumor biopsies.

Table 1 below outlines exemplary steps involved to go from E. coli toDNA suitable for sequencing. In some instances, conventional methodsrequire centrifugation, several temperatures, wash steps, and numeroustransfer steps which are inefficient. In contrast, as described herein,in some embodiments allows the same steps to be carried out by a devicethat minimizes the pipette transfers and exposure to large virginsurfaces with varying degrees of nonspecific binding properties. In someinstances, the device is temperature controlled to provide appropriatereaction conditions. In some instances, PCR, cycle PCR for sequencingpre-amp or full PCR (endpoint, real time or digital) is accomplishedoff-device or on-device. Off-device includes not on the device but onthe same cartridge assembly, connections via fluidic channels orconduits. Furthermore, in some instances PCR amplification isaccomplished in the device flow cell chamber, in a PCR tube that is onthe cartridge, or though fluidic channels that possess heat zones fortemperature cycling. In some instances, the eluate from the devicechamber is combined with side channel(s) primed with non aqueousmiscible fluid, e.g., oil, and other droplet stabilizers to performamplification in droplets. In some embodiments, the temperature cyclingmechanics are as described above.

In Table 1, the amount of starting material for the conventionalprocessing was 2-5×10⁷ E. Coli cells in approximately 1 ml of water andthe entire amount was concentrated on the filters. Using the chip, asdisclosed herein, only 1×10⁴ E. Coli cells in approximately 50microliters was applied to the flow cell. This was 3 orders of magnitudeless starting material.

TABLE 1 Comparison of methods for nucleic acid isolation. ConventionalAn exemplary embodiment provided herein Epicentre WaterMaster DNAPurification Kit [ON CHIP] Concentrate E. coli bacteria on filtersCapture E. coli, 1 MHz, 10 Vpp, 10′ Lysis solution Electro-lyse E. coli,200 V DC, 1 msec pulse Proteinase K treatment, 65 C., 15′ De-energizeelectrodes RNase treatment, 37 C., 30′ Collection, 10 Vpp, 10 KHzPrecipitate protein by centrifuge, 10 K x g, 4 C., De-energizeelectrodes 10′ Wash isopropanol Q protease treatment, 37 C., 10′Precipitate protein by centrifuge, 10 K x g, 4 C., Inactivate 70 C., 10′10′ Rinse pellet with 70 ethanol Collection, 10 Vpp, 10 KHz ResuspendDNA in TE buffer Wash with Nextera LMW buffer Remove inhibitors, 2 K xg, 2′ Repeat 2X DNA ready for library prep Epicentre Nextera DNA SamplePrep (Illumina) 50 ng input DNA (1e7 E. coli equiv.) Fragment withTransposase, 55 C., 5′ Fragment with Transposase, 55 C., 7′ Purify withZymo spin column, 10 K x g, 1′ Elute DNA into microtube Add adapters,cycle PCR, 9 cycles [OFF CHIP] Purify with Zymo spin column, 10 K x g,1′ Add adapters, cycle PCR, 9 cycles Purify with Zymo spin column, 10 Kx g, 1′ Sequence Sequence

In various embodiments (i.e., depending on the AC electrokineticparameters), cells or other micron scale particles are concentrated toeither the low or high field regions. In some instances, the crossoverfrequency which determines whether a particle moves into or away fromthe high field region can be tuned by varying the AC frequency, voltage,medium conductivity, adulterating particle polarizability (such asattachment or binding of materials with different DEP characteristics),or electrode geometry. In some instances, nanoscale particles arelimited to concentration in the high field region. In some instances,Brownian motion and other hydrodynamic forces limit ability toconcentrate in low field regions.

Detection and Characterization of Cancer Using Cell Free Biomarkers

Assays may be performed on circulating cell-free high molecular weightDNA (>300 bp) and other target cell-free biomarkers isolated using themethods and devices disclosed herein to characterize cancer in patientsusing target specific cell-free biomarkers. “Characterization” of cancerincludes but is not limited to detection and diagnosis of cancer,prognosis of disease, treatment response monitoring and other actionsrelated to cancer analysis and treatment therein.

In some embodiments, the characterization may be performed via molecularprofiling of cell free biomarkers. The profiling includes but is notlimited to enumeration of analytes, specific detection of analytes,including but not limited to proteins, lipids, antibodies, tumor DNA,tumor cells, exosomes, nucleosomes, nanosomes detection of specific genesequences, detection of mutant gene sequences, detection of loss ofheterozygosity, determination of methylation status, detection ofalterations, detection of deletions and other molecular profiling assaysused in the analysis and characterization of physical and/or biochemicalstatus of a patient or subject.

Cell free biomarkers can be derived from proteins or moleculesassociated with cellular exocytosis, necrosis, or secretion processes.Markers include: high molecular weight dna (>500 bp), nucleosomes,exosomes, aggregated proteins, cell membrane fragments, mitochondria,cellular vesicles, and other markers related to cellular exocytosis,necrosis or secretion.

Examples of candidates for circulating cell-free biomarkers include butare not limited to circulating tumor DNA, including mutations ordeletions, rearrangement, methylated nucleic acid, loss ofheterozygosity, and other DNA alterations. RNA may also be used,including micro RNA (miRNA), RNA from microvesicles and other RNA formsthat provide useful information with regards to the characterization of,for example, cancer diagnosis, prognosis and treatment response in apatient. Tumor cells may also be directly monitored, as well as cellfree proteins, including but not limited to GFAP, VEGF, EGFR, b-FGF,KRAS, YKL-40 and MMP-9.

The methods and devices disclosed herein for characterization of, forexample, cancer patients and subjects uses AC Electrokinetics to isolatecell free target biomarkers directly from whole blood, serum, plasma, orother bodily fluid or sample. The methods and devices disclosed hereinuses minimal amounts of sample, for example, up to 10 μl, up to 20 μl,up to 30 μl, up to 40 μl, up to 50 μl, up to 60 μl, up to 70 μl, up to80 μl, up to 90 μl, up to 100 μl, up to 200 μl, up to 300 μl, up to 400μl, up to 500 μl or more of sample. In some embodiments, the methods anddevices disclosed herein uses less than 500 μl, less than 400 μl, lessthan 300 μl, less than 200 μl μl, less than 100 μl, less than 90 μl,less than 80 μl, less than 70 μl, less 60 μl, less than 50 μl, less than40 μl, less than 30 μl, less than 20 μl, less than 10 μl or less than 5μl of sample. In some embodiments, the methods and devices disclosedherein use between about 50 μl of sample and about 500 μl of sample.

The methods and devices disclosed herein for characterization of, forexample, cancer patients and subjects may use intercalating dyes,antibody labeling, or other traditional staining techniques to enabledirect quantification using fluorescence microscopy or other detectiontechniques. The methods and devices disclosed herein may also useDNA/RNA hybridization techniques to detect specific alleles implicatedin cancer. The methods and devices disclosed herein may also useQuantitative Real Time PCR, including of nuclear or mitochondrial DNA orother target nucleic acid molecule markers, enzyme-linked immunosorbentassays (ELISA), direct SYBR gold assays, direct PicoGreen assays, lossof heterozygosity (LOH) of microsatellite markers, optionally followedby electrophoresis analysis, including but not limited to capillaryelectrophoresis analysis, sequencing and/or cloning, including nextgeneration sequencing, methylation analysis, including but not limitedto modified semi-nested or nested methylation specific PCR, DNA specificPCR (MSP), quantification of minute amounts of DNA after bisulfitomeamplification (qMAMBRA), as well as methylation on beads, mass-basedanalysis, including but not limited to MALDI-ToF (matrix-assisted laserdesorption/ionization time of flight analysis, optionally in combinationwith PCR, and digital PCR.

The methods and devices disclosed herein may employ dyes, includingintercalating dyes, antibody labeling, stains and other imagingmolecules that enable direct quantification of the cell-free biomarkermaterials directly on or in use with the embodied devices, including theuse of fluorescence microscopy. Examples of fluorescent labeling ofnucleic acids, e.g. DNA and RNA, include but are not limited to cyaninedimers high-affinity stains (Life Technologies) can used. Among themYOYO®-1, YOYO®-3, POPO™-1, POPO™-3, TOTO®-1, TOTO®-3 are the preferredstaining dyes. Fluorescent labeling of protein for detection andquantitation in conjunction with the methods and devices disclosedherein include but not limited to Quanti-iT™ protein quantitation assay,NanoOrange™ protein quantitation assay, CBQCA protein quantitation assay(Life Technologies). Fluorescent quantitation of other cancer biomarkersmay also be used including mitochondria, labelling dyes such asMitoTracker® Green FM® and MitoTracker® Red FM®.

The methods and devices disclosed herein may also be used in conjunctionwith DNA/RNA hybridization techniques to detect specific allelesimplicated in cancer. In some embodiments, specific electrodes andcorresponding electrode trace lines can be designed to individuallycontrol separate electrode so as to achieve a unique electric fielddistribution. By designing nonuniform electric field distribution,specific DNA/RNA can be manipulated.

Additionally, the microelectrode arrays disclosed herein may be furtherfunctionalized, for example, by covering the array with a reactivehydrogel. The hydrogel may comprise binding partners, including biotinbinding protein; alternatively, the hydrogel may also be functionalizedby acylation or by surface modification to chemisorb oligonucleotides onthe surface. The methods and devices disclosed herein may further bemanipulated to attain control of hybridization and detection of specificalleles, for example, through the use of a Complimentary Metal-OxideSemiconductor (CMOS) device that can control the microelectrode array ina manner that allows for multiple use of the array and high-throughputscreening of matching oligonucleotides.

The methods and devices disclosed herein are also capable of elutingcirculating cell-free target biomarkers such as nucleosomes, highmolecular weight DNA, exosomes and proteins for post-genetic analysisand for quantification and further analysis using quantitative PCR,reverse transcriptase (RT) PCR, and sequencing analytical techniques foridentifying proteins or nucleic acids of interest in the isolated andeluted sample DNA. Post-genetic analysis is performed on nucleosomal ornucleoprotein complexed dsDNA (greater than 300 bp), on exosomal dsDNAor RNA (greater than 100 bp), and/or on mitochondrial DNA.

Candidates of cell-free biomarkers (ccfDNA=circulating cell-free DNA)for detecting cancer using the methods and devices disclosed hereininclude the following:

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Patients with higher RASSF1Aconcentrations at diagnosis or one year after tumor resection showedpoorer disease-free survival. The circulating concentration of RASSF1Ain HBV carriers increased significantly from the time of enrollment tocancer diagnosis Microsatellite instability and loss of heterozygosityof D8S277, D8S298, and D8S1771 at chromosome 8p were detected on theplasma DNA of HCC patients. Pancreatic Methylation profile of MelnikovAA. J Surg Oncol. 2009 Feb Carcinoma circulating plasma DNA in 1; 99(2):119-22. patients with pancreatic Gornik I. Clin Biochem. 2009 Jan;cancer. 42(1-2): 38-43. Free serum DNA is an early Sawabu N. Pancreas.2004 Apr; predictor of severity in acute 28(3): 263-7. pancreatitis.Liggett T. Cancer. 2010 Apr 1; Differential methylation of 116(7):1674-80. cell-free circulating DNA among patients with pancreatic cancerversus chronic pancreatitis. Gastrointestinal Esophageal Quantificationof circulating Tomita H. Anticancer Res. 2007 Jul- Tract Tumors plasmaDNA revealed that Aug; 27(4C): 2737-41. up to 61% of patients withHoffmann AC. J Cancer Res Clin esophageal carcinoma have Oncol. 2009Sep; 135(9): 1231-7. detectable levels of methylated DAPK (Death-associated protein kinase) or APC (adenomatous polyposis coli gene)promoter DNA and are associated with unfavorable prognosis Stoch TumorsPlasma DNA concentration is Kolesnikova EV. Ann N Y Acad Sci. higher inpatients with 2008 Aug; 1137( ): 226-31. gastric cancer compared Tani N.2006; 33: 1720-1722. with controls. Sai S. Anticancer Res. 2007 Jul-Aug;Epigenetic changes of cell- 27(4C): 2747-51. free serum DNA of RUNX3,Sakakura C. Anticancer Res. 2009 MGMT, p15, and hMLH1 Jul; 29(7):2619-25. hypermethylation in postoperative monitoring. methylationstatus of CEA, P16, E-cadherin, RARbeta and CDH4 genes Colorectal Morethan half of the Schwarzenbach H. Ann N Y Acad Sci. Tumors patients withearly stage 2008 Aug; 1137( ): 190-6. disease contain mutant DNA daSilva Filho BF. J Clin Pathol. 2013 in their circulation. Sep; 66(9):775-8. Mutated circulating DNA Lofton-Day C. Clin. Chem. may depend ontumor 2008; 54: 414-423. clonality Tóth K. Orv Hetil. 2009 May 24;Aberrant methylation status 150(21): 969-77. of specific genes such asdeVos T. Orv Hetil. 2009 May 24; SEPT9, HPP1 and/or HLTF in 150(21):969-77. the serum of patients with deVos T. Clin Chem. 2009 Jul;colorectal cancer has the 55(7): 1337-46. potential to become a pre-Wallner M. Clin Cancer Res. 2006 therapeutic predictor of Dec 15;12(24): 7347-52. outcome. Misale S. Nature. 2012 Jun 28; Studies havesuggested that 486(7404): 532-6. the emergence of KRAS Trevisiol C. IntJ Biol Markers. 2006 mutant clones can be Oct-Dec; 21(4): 223-8.detected non-invasively Umetani N. Clin Chem. 2006 Jun; months beforeradiographic 52(6): 1062-9. progression and can be used Morgan SR. JClin Pathol. 2013 Sep; for monitoring of drug 66(9): 775-8. resistance.Mouliere F. Transl Oncol. 2013 Jun; Serum DNA integrity 6(3): 319-28.increased in CRC in localized Trevisiol C. Int J Biol Markers. 2006lesions and in advanced Oct-Dec; 21(4): 223-8. stage cancer. Nakayama G.Anticancer Res. Early stage detection of 2007; 27: 1459-1463. tumorincreased when CCFDNA was used in combination with CEA measurement.Other mutations include microsatellite instability, BRAF, and SMAD4 andabnormal promoter methylation including TMEFF2, NGFR, and p16 have beendetected in ccfDNA. Head and Neck Nasopharyngeal Aberranthypermethylated Chan KC. Clin Cancer Res. 2008 Jul 1; Tumors Carcinomapromoter DNA of at least 14(13): 4141-5. one of the five following JiangWW. Int J Cancer. 2006 Dec 1; genes; CDH1, p16, DAPK1, 119(11): 2673-6.p15, and RASSF1A was Chan SL. BMC Cancer. 2006 Oct 31; detectable in 71%of plasma 6( ): 259. of NPC patients before Chan KC. Semin Cancer Biol.2002 treatment. Dec; 12(6): 489-96. Hypermethylation of the promoter DNAof at least one in three genes (CDH1, DAPK1, and p16) was detectable inthe post- treatment plasma of 38% of recurrent NPC patients and none ofthe patients in remission. EBV-DNA as a sensitive and specific marker inmonitoring NPC by detecting early recurrence and correlation withtreatment response. Thyroid Detection of free circulating Chuang TC. eadNeck. 2010 Feb; Tumors mutant BRAF/DNA in 32(2): 229-34. patients withpapillary thyroid carcinoma (PTC) Lymphoma/ Higher levels of CCFDNA inHohaus S. Ann Oncol. 2009 Aug; Leukemia patients with Hodgkin 20(8):1408-13. lymphoma (HL), diffuse Jiang Y. 2012 Feb; 20(1): 53-6. large Bcell Lymphoma (DLBCL), and mantle cell lymphoma than in healthyindividuals and were associated with advanced stage disease, presence ofB- symptoms, elevated lactate dehydrogenase levels, and age >60 years.Rearranged immunoglobulin heavy chain DNA has been found in the plasmaof patients with non-Hodgkin's lymphoma and acute B cell leukemia. MGMTpromoter hypermethylation along with p53 mutation as useful prognosticmarkers in diffuse large B cell Lymphoma (DLBCL). Lung Cancer Non SmallCell Plasma DNA levels are Cheng C. Cancer Sci. 2009 Feb; Lung increasedin lung cancer 100(2): 303-9. Carcinoma patients compared to normal XieGS. Chin. Med. J. 2004; 117: 1485-1488. (NSCLC) healthy controls andhigher Yoon KA. J Mol Diagn. 2009 May; concentration has been 11(3):182-5. associated with poor Lee SM. Mol Cells. 2012 Aug; prognosis.34(2): 171-6. Complete or partial post Ponomaryova AA. Lung Cancer.treatment response to 2013 Sep; 81(3): 397-403. chemotherapy alsoNakamura T. J Thorac Oncol. 2012 correlates with no mutation Sep; 7(9):1369-81. detection. Methylation status of 14-3-3 sigma of serum DNA inpretreatment condition and for P16M in pleural lavage were associatedwith survival. Hypermethylation of RASSF1A, p14 (ARF) and APC are usefulprognostic markers in patients receiving gemcitabine, and testing plasmaDNA for K- RAS mutation is helpful in monitoring NSCLC patientsreceiving paclitaxel and carboplatin. Detection of epidermal growthfactor receptor (EGFR) mutations using plasma DNA as essential todetermine appropriate lung cancer treatment and monitoring Small CellLung Microsatellite markers or Board RE. Ann. N. Y. Acad. Sci. CarcinomaLOH are useful for the 2008; 1137: 98-107. (SCLC) detection ofalterations in Tamkovich SN. Ann N Y Acad Sci. the plasma DNA of SCLC2008 Aug; 1137( ): 214-7. patients. Male Genital Testicular IncreasedCCFDNA and cell- Ellinger J. J Urol. 2009 Jan; Tract Tumors free serummtDNA (79-bp 181(1): 363-71. (mtDNA-79) and 220 bp Ellinger J. J Urol.2009 Jul; (mtDNA-220)) levels levels 182(1): 324-9. in testicular tumorsand correlation with tumor stage. Hypermethylation of CCFDNA APC, GSTP1,PTGS2, p14 (ARF), p16 (INK) and RASSF1A as potential biomarkers fordetection and monitoring Urinary System Kidney Tumors Tumor DNA fromrenal cell Goessl C. Eur Urol. 2002 Jun; carcinoma, bladder cancer,41(6): 668-76. or prostate cancer could be Cairns P. Ann N Y Acad Sci.2004 Jun; detectable in more than 50% 1022( ): 40-3. of plasma/serum andin more than 70% of urine samples Promoter hypermethylation of ccfDNAfor VHL, p16/CDKN2a, p14ARF, APC, RASSF1A, and Timp-3 detected with 88%sensitivity and almost 100% specificity. Prostate CCFDNA and PSA assaygave Delgado PO. Tumour Biol. 2013 Apr; Carcinoma 89% sensitivity indetecting 34(2): 983-6. (PCa) PCa Chun FK BJU Int. 2006 Sep; Combinationof DNA load 98(3): 544-8. and promoter methylation Papadopoulou E. Ann NY Acad Sci. status identified 88% of PCa. 2006 Sep; 10750: 235-43. LOHand genetic aberrations Muller I. Clin Chem. 2008 Apr; such as allelicimbalance (AI) 54(4): 688-96. and epigenetic changes of Ellinger J. IntJ Cancer. 2008 Jan 1; promoter hypermethylation 122(1): 138-43.(methylation of RASSF1, Cortese R. Hum Mol Genet. 2012 Aug RARB2, andGSTP1) have 15; 21(16): 3619-31. also been detected in CCFDNA ofprostate cancer patients Significant associations between LOH andincreasing Gleason scores for the marker combinations of D6S1631,D8S286, D9S171, D8S286 and D9S171. Methylation of the GSTP1 gene foundin 25% of free plasma DNA and in 94% of tissue samples. Concentrationsof apoptotic PTGS2 fragments discriminate sensitivity (88%) andspecificity (64%) between BPH (benign prostate hypertrophy) and PCa butthe apoptotic index (AI) was more specific (82%) but less sensitive(70%) Skin Malignant Higher levels of CCFDNA in Daniotti M. Int JCancer. 2007 Jun 1; Melanoma patients compared to 120(11): 2439-44.controls and can be helpful Nakamoto D. Bull Tokyo Dent Coll. inmonitoring the disease in 2008 May; 49(2): 77-87. late stage (stage IV)but Lo Nigro C. J Invest Dermatol. 2013 unsatisfactory for the earlyMay; 133(5): 1278-85. detection of melanoma LOH at microsatellitemarkers D1S243, D6S311, D9S161 and D19S246 in the plasma is alsoassociated with malignant mucosal melanoma (MMM) and it could be usefulmarker for diagnosis of recurrence and metastasis MMM Detection ofTFPI2- methylated DNA in the serum of patients with resected melanoma asa sensitive and specific biomarker of recurrence or metastatic melanomaSquamous Cell In 90% of squamous cell Kakimoto Y. Oncol Rep. 2008 Nov;Carcinoma carcinomas of the oral 20(5): 1195-200. cavity, there is amicrosatellite alteration in serum DNA that is identical to those in thecorresponding tumor DNA which may provide valuable prognosticinformation and serve as a guide for future therapy. AdditionalReferences include: Holdhoff et al. J. Neurooncol. 2013, 113, 345;Elshimali et al. Int. J. Mol. Sci. 2013, 14, 18925; Swanson et al.Senson Actuat. B-Chem. 2000, 64, 22; Sosnowski et al. Proc. Natl. Acad.Sci. U.S. 1997, 94, 1119; Huang et al. Macromolecules 2002, 35, 1175;and Hofman et al. RSC Advances 2012, 2, 3885.

DEFINITIONS AND ABBREVIATIONS

The articles “a”, “an” and “the” are non-limiting. For example, “themethod” includes the broadest definition of the meaning of the phrase,which can be more than one method.

“Vp-p” is the peak-to-peak voltage.

“TBE” is a buffer solution containing a mixture of Tris base, boric acidand EDTA.

“TE” is a buffer solution containing a mixture of Tris base and EDTA.

“L-Histidine buffer” is a solution containing L-histidine.

“DEP” is an abbreviation for dielectrophoresis.

“ACE” is Alternating Current Electrokinetics

EXAMPLES Example 1 Formation of Hydrogel by Spin-Coating (Two Coats) 1

For a layer of hydrogel, approximately 70 microliters of hydrogel isused to coat a 10×12 mm chip.

A low concentration (≦1% solids by volume) cellulose acetate solution isdissolved into a solvent such as acetone, or an acetone and ethanolmixture and applied to an electrode array chip as disclosed herein. Thechip is spun at a low rpm rate (1000-3000). The low rpm rate ensuresthat the height of the gel is in the range of 500 nm or greater.

The first (bottom) coating of cellulose acetate is dried at roomtemperature, in a convection oven, or a vacuum oven. Optionally, thesecond layer of cellulose-acetate spin-coat is added immediately.

The second layer of cellulose acetate comprises a high concentration(≧2%) of cellulose acetate dissolved into a solvent such as acetone, oran acetone and ethanol mixture. After a second layer of celluloseacetate is added, the chip is spun at a high rpm rate (9000-12000). Thehigh rpm rate will ensure the height of the gel is in the range of 300nm or less.

The chip with two layers of cellulose acetate is then dried at roomtemperature, in a convection oven, or in a vacuum oven.

Example 2 Formation of Hydrogel with Additives by Spin-Coating (TwoCoats)

For a layer of hydrogel, approximately 70 microliters of hydrogel isused to coat a 10×12 mm chip.

A low concentration (≦1% solids by volume) cellulose acetate solution isdissolved into a solvent such as acetone, or an acetone and ethanolmixture and applied to an electrode array chip as disclosed herein. Thechip is spun at a low rpm rate (1000-3000). The low rpm rate ensuresthat the height of the gel is in the range of 500 nm or greater.

The first (bottom) coating of cellulose acetate is dried at roomtemperature, in a convection oven, or a vacuum oven. Optionally, thesecond layer of cellulose-acetate spin-coat is added immediately.

The second layer of cellulose acetate comprises a high concentration(≧2%) of cellulose acetate dissolved into a solvent such as acetone, oran acetone and ethanol mixture. A low concentration (1-15%) ofconductive polymer (PEDOT:PSS or similar) is added into the secondcellulose acetate solution. After a second layer of cellulose acetate isadded, the chip is spun at a high rpm rate (9000-12000). The high rpmrate will ensure the height of the gel is in the range of 300 nm orless.

The chip with two layers of cellulose acetate is then dried at roomtemperature, in a convection oven, or in a vacuum oven.

Example 3 Formation of Hydrogel with Additives by Spin-Coating (ThreeCoats)

For a layer of hydrogel, approximately 70 microliters of hydrogel isused to coat a 10×12 mm chip.

A low concentration (≦1% solids by volume) cellulose acetate solution isdissolved into a solvent such as acetone, or an acetone and ethanolmixture and applied to an electrode array chip as disclosed herein. Thechip is spun at a high rpm rate (9000-12000). The low rpm rate ensuresthat the height of the gel is in the range of 300 nm or less.

The first (bottom) coating of cellulose acetate is dried at roomtemperature, in a convection oven, or a vacuum oven. Optionally, thesecond layer of cellulose-acetate spin-coat is added immediately.

The second layer of cellulose acetate comprises a high concentration(≧2%) of cellulose acetate dissolved into a solvent such as acetone, oran acetone and ethanol mixture. A low concentration (1-15%) ofconductive polymer (PEDOT:PSS or similar) is added into the secondcellulose acetate solution. After a second layer of cellulose acetate isadded, the chip is spun at a low rpm rate (1000-3000). The low rpm ratewill ensure that the height of the gel is in the range of 500 nm orgreater.

The second coating of cellulose acetate is dried at room temperature, ina convection oven, or a vacuum oven. Optionally, the third layer ofcellulose-acetate spin-coat is added immediately.

The third layer of cellulose acetate comprises a high concentration(≧2%) of cellulose acetate dissolved into a solvent such as Acetone, oran Acetone Ethanol mixture. The chip is spun at a high rpm rate(9000-12000). The low rpm rate ensures that the height of the gel is inthe range of 300 nm or less.

The chip with three layers of cellulose acetate is then dried at roomtemperature, in a convection oven, or in a vacuum oven.

Example 4 Chip Construction

For FIGS. 2 & 3: A 45×20 custom 80 μm diameter circular platinummicroelectrode array on 200 um center-center pitch was fabricated basedupon previous results (see references 1-3, below). All 900microelectrodes are activated together and AC biased to form acheckerboard field geometry. The positive DEP regions occur directlyover microelectrodes, and negative low field regions occur betweenmicroelectrodes. The array is over-coated with a 200 nm-500 nm thickporous poly-Hema hydrogel layer (Procedure: 12% pHema in ethanol stocksolution, purchased from PolySciences Inc., that is diluted to 5% usingethanol. 70 uL of the 5% solution is spun on the above mentioned chip ata 6K RPM spin speed using a spin coater. The chip+hydrogel layer is thenput in a 60° C. oven for 45 minutes) and enclosed in a microfluidiccartridge, forming a 50 μl sample chamber covered with an acrylic window(FIG. 1). Electrical connections to microelectrodes are accessed fromMolex connectors from the PCB board in the flow cell. A functiongenerator (HP 3245A) provided sinusoidal electrical signal at 10 KHz and10-14V peak-peak, depending on solution conductivity. Images werecaptured with a fluorescent microscope (Leica) and an EGFP cube (485 nmemission and 525 nm excitation bandpass filters). The excitation sourcewas a PhotoFluor II 200 W Hg arc lamp.

-   [1] R. Krishnan, B. D. Sullivan, R. L. Mifflin, S. C. Esener,    and M. J. Heller, “Alternating current electrokinetic separation and    detection of DNA nanoparticles in high-conductance solutions.”    Electrophoresis, vol. 29, pages 1765-1774, 2008.-   [2] R. Krishnan and M. J. Heller, “An AC electrokinetic method for    enhanced detection of DNA nanoparticles.” J. Biophotonics, vol. 2,    pages 253-261, 2009.-   [3] R. Krishnan, D. A. Dehlinger, G. J. Gemmen, R. L. Mifflin, S. C.    Esener, and M. J. Heller, “Interaction of nanoparticles at the DEP    microelectrode interface under high conductance conditions”    Electrochem. Comm., vol. 11, pages 1661-1666, 2009.

Example 5 Isolation of Human Genomic DNA

Human Genomic DNA (gDNA) was purchased from Promega (Promega, Madison,Wis.) and was sized to 20-40 kbp. (Sizing gel not shown.) The gDNA wasdiluted in DI water to the following concentrations: 50 nanograms, 5nanograms, 1 nanogram, and 50 picograms. The gDNA was stained using1×SYBR Green I green fluorescent double stranded DNA dye purchased fromInvitrogen (Life Technologies, Carlsbad, Calif.). This mixture was theninserted into the microelectrode arrays and run at 14 Volts peak to peak(Vp-p), at 10 kHz sine wave for 1 minute. At the conclusion of 1 minute,a picture of the microelectrode pads was taken using a CCD camera with a10× objective on a microscope using green fluorescence filters (FITC) sothat the gDNA could be visualized (FIG. 2) The chip was able to identifydown to 50 pg of gDNA in 50 μL water, i.e. 1 ng/mL concentration.Additionally, at 50 picograms, each microelectrode had on average ˜60femtograms of DNA since there are 900 microelectrodes on the array. Thelow-level concentration ability of the ACE device is well within therange of 1-10 ng/mL needed to identify Cfc-DNA biomarkers in plasma andserum (see references 4-6 below).

-   [4] T. L. Wu et al, “Cell-free DNA: measurement in various    carcinomas and establishment of normal reference range.” Clin Chim    Acta., vol. 21, pages 77-87, 2002.-   [5] R. E. Board et al, “DNA Methylation in Circulating Tumour DNA as    a Biomarker for Cancer”, Biomarker Insights, vol. 2, pages 307-319,    2007.-   [6] O. Gautschi et al, “Circulating deoxyribonucleic Acid as    prognostic marker in non-small-cell lung cancer patients undergoing    chemotherapy.” J Clin Oncol., vol. 22, pages 4157-4164, 2004.

Example 6 Isolation of DNA from E. Coli

Using the Chip and methods described in Examples 4 and 5, approximately5000 green fluorescent E. coli cells in 50 uL of fluid was inserted intoa chip and run using protocol described in caption for FIG. 3. Panel (A)shows a bright field view. Panel (B) shows a green fluorescent view ofthe electrodes before DEP activation. Panel (C) shows E. coli on theelectrodes after one minute at 10 kHz, 20 Vp-p in 1×TBE buffer. Panel(D) shows E. coli on the electrodes after one minute at 1 MHz, 20 Vp-pin 1×TBE buffer.

The E. coli depicted in FIG. 3 were lysed using a 100 milli-second 100VDC pulse using the HP 3245A function generator. The lysed particulateswere then gathered on the electrode surface using 10 kHz, 10Vp-p and theIllumina Nextera Protocol was used for library prep for sequencing whilethe DNA was on the chip (by inserting the appropriate buffers at theappropriate times onto the chip) to tag the DNA for Sequencing. The DNAwas then eluted in 50 uL of 1×TBE Buffer and then PCR amplified for 9-12cycles (using the Nextera Protocol) on a Bio-Rad PCR machine. Theamplified DNA was then run on an Illumina GA II Sequencer. DNA from E.Coli was also isolated from 1×TBE buffer (10 million cells) using theEpicentre™ WaterMaster™ DNA purification procedure, to serve as a goldstandard for comparison. The results are depicted in FIG. 4.

Example 7 Formation of Hydrogel with GVD

Hydrogel, such as polyhydroxyethylmethacrylate (pHEMA) may also belayered onto the chip surface via vapor deposition using proprietaryassays developed by GVD Corporation (Cambridge, Mass.) (seewww.gvdcorp.com). Hydrogels such as pHEMA were deposited in variousthickness (100, 200, 300, 400 nm) and crosslinking (5, 25, 40%) densityon electrode chips was performed using technology developed by GVDCorporation. The hydrogel films were tested using a standard ACEprotocol (no pretreatment, 7Vp-p, 10 KHz, 2 minutes, 0.5×PBS, 500 ng/mlgDNA labeled with Sybr Green 1). Fluorescence on the electrodes wascaptured by imaging. FIG. 10 shows that 100 nm thickness, 5% crosslinkgel device was found to have strong DNA capture. The process could alsobe optimized by changing the deposition rate or anchoring growth to thesurface of the microelectrode array (i.e., to the passivation layer andexposed electrodes), using an adhesion promoter such as a silanederivative.

Example 8 Performance of Disclosed Device and Method v. ConventionalMethod

QIAGEN® circulating nucleic acid Purification kit (cat#55114) was usedto purify 1 ml of plasma from chronic lymphocytic leukemia (CLL)patients, according to manufacturer's protocol. Briefly, incubation of 1ml plasma with Proteinase K solution was performed for 30 minutes at 60°C. The reaction was quenched on ice and the entire volume was applied toa QIAamp Mini column connected to a vacuum. The liquid was pulledthrough the column and washed with 3 different buffers (600-750 uleach). The column was centrifuged at 20,000×g, 3 minutes and baked at56° C. for 10 minutes to remove excess liquid. The sample was eluted in55 μl of elution buffer with 20,000×g, 1 minute centrifugation. Totalprocessing time was ˜2.5 hours.

The chip die size was 10×12 mm, with 60-80 μm diameter Pt electrodes on180-200 μm center-to-center pitch, respectively. The array wasovercoated with a 5% pHEMA hydrogel layer (spun cast 6000 rpm fromEthanol solution, 12% pHEMA stock from Polysciences). The chip waspretreated using 0.5×PBS, 2V rms, 5 Hz, 15 seconds. The buffer wasremoved and 25 μl of CLL patient plasma was added. DNA was isolated for3 minutes at 11 V p-p, 10 Khz, then washed with 500 μl of TE buffer at a100 μl/min flow rate, with power ON. The voltage was turned off and theflow cell volume was eluted into a microcentrifuge tube. Totalprocessing time was ˜10 minutes.

The same process can be applied to fresh whole blood withoutmodification. Ability to extract and purify DNA from whole undilutedblood is uniquely enabled by the chip technology disclosed herein.

DNA quantitation was performed on the Qiagen and chip elutes usingPicoGreen according to manufacturer's protocol (Life Tech) (Table 2).

Subsequent gel electrophoresis, PCR and Sanger sequencing reactionsshowed similar performance for both extraction techniques with the chipbeing able to process whole blood as well as plasma. Mann-Whitney Unon-parametric statistical test was also run between DNA amountsisolated from plasma using the Qiagen and chip techniques. There was nostatistical difference (p<0.05 two-tailed) using either method of DNApurification.

TABLE 2 DNA purification, chip v. Qiagen Values are in ng/ml andnormalized to original plasma sample volume for comparison purposes.Chip- Qiagen- Chip- Patient plasma plasma blood normal A 139 39 274normal B 206 80 114 normal C 133 32 97 TJK 528 320 547 167 TJK 851 218393 307 TJK 1044 285 424 794 TJK 334 261 1387 666 TJK 613 179 53 257 TJK762 145 367 314 TJK 847 886 1432 811 TJK 248 84 119 448 TJK 1024 302 169332 TJK 1206 584 396 1435 TJK 1217 496 146 584 TJK 1262 87 84 1592 TJK1311 119 257 1825

Example 9 On-Chip Quantification Using Labeling and FluorescenceMicroscopy

Using ACE microfluidic cartridges relative concentration of cell freebiomarkers were determined in an unknown sample (sample can be wholeblood, serum, plasma). The ACE microfluidic cartridge may be designedwith one or more chambers for known standards and one or more chambersfor the unknown sample as shown in FIG. 11.

An ACE field at 10 Vp-p and 10 kHz is applied to select microfluidiccartridge chambers and cell-free target biomarkers of interest arecaptured on the electrodes as shown in FIG. 12. A fluidic wash solution(water+osmolytes) is applied to wash away unwanted sample, i.e.particles and other components that are not captured on the electrodes.This fluidic wash is compatible with polymerase chain reaction (PCR) andnext-generation sequencing thus allowing for secondary analysispost-elution.

Target biomarkers include proteins, lipids, antibodies, high molecularweight DNA (greater than 300 bp), tumor cells, exosomes, nucleosomes,nanosomes. For the fluorescent detection/quantification of suchbiomarkers specific dyes use such as YOYO®-1, SYBR® Green, CBQCA proteinquantitation kit, SYTO® RNASelect™.

Once excess unwanted sample is washed away, a CCD/CMOS/PMT detector isused in conjunction with fluorescence microscopy (with appropriateexcitation/emission filters) to enable direct detection (binary) and/orquantification (concentration) of unknown analytes using aRegion-of-Interest (ROI) image segmentation algorithm that comparedpixel intensity between two regions in the electrodes (FIG. 12).Fluorescent quantification form both the known and unknown chambers isdetermined and the data is then compared using a linear fit calibrationcurve (FIG. 13) to create a relative ratio of intensity between theknown chamber and the unknown chamber. Because the analytes in the knownchamber have known specific concentrations of the cell free targetbiomarkers and the fluorescence labels are specific for the targetanalytes, using an algebraic relationship between intensities of theknown chamber and the unknown chamber enable and the linear calibrationcurve the determination of analyte concentration from the unknownchamber.

Example 10 Off-Chip Quantification using Q-PCR, RT-PCR and Sequencing

Using ACE microfluidic cartridges, relative concentrations of cell-freebiomarkers were determined in an unknown sample. The sample may be wholeblood, serum, plasma or other biological sample/fluid. The ACEmicrofluidic cartridge may be designed with one or more chambers forknown standards and one or more chambers for the unknown sample.

An ACE field at 10 Vp-p and 10 kHz is applied to the chamber andcell-free target biomarkers of interest are captured on the electrodes(FIG. 12). A fluidic wash (water+osmolytes) is applied using aperistaltic pump while the ACE field is still on in order to remove allunwanted sample. This fluidic wash is compatible with polymerase chainreaction (PCR) and next-generation sequencing thus allowing forsecondary analysis post-elution.

The ACE field is turned off and the captured particles are released intothe PCR/Sequencing compatible solution. The unknown sample is elutedfrom the electrodes, and the sample used in PCR or next-gen sequencinganalysis to determine specific gene sequences, alterations or deletionsthat may be present in the eluted dsDNA or RNA. FIG. 14 illustrates theusage of PCR technique for detection of PCR mutations for DNA samplesfrom the cell lines H1975, RKO, OCI-AML3 and HEL 92.1.7 diluted ineither H₂O or ACE fluidic wash solution and used as positive controlsfor EGFR T790, BRAF V600, NPM1a and JAK2 617V assays.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method for quantifying nucleic acid in a sample, the methodcomprising: a. applying a sample with a conductivity of greater than 100mS/m to a device, the device comprising an array of electrodes capableof establishing an AC electrokinetic field region, the device furthercomprising at least two chambers, the sample applied to a first chamber;b. applying known quantities of a nucleic acid standard to a secondchamber; c. establishing a first AC electrokinetic high field region inthe first chamber, the first AC electrokinetic high field capable ofisolating larger nanoparticulate molecular targets; d. establishing asecond AC electrokinetic low field in the first chamber, the second ACelectrokinetic low field capable of concentrating cells or micron-sizedentities that may be present in the sample; e. establishing a third ACelectrokinetic high field to the second chamber, the second ACelectrokinetic high field isolating the molecular nucleic acid standardapplied to the second chamber; f. flushing cells and micron-sizedentities that may be present in the sample from the first chamber; g.detecting bound nucleic acid signal on the array in the first chamberand second chamber; and h. quantifying the bound nucleic acid bycomparing the detected signal from the first chamber to detected signalfrom the second chamber.
 2. The method of claim 1, wherein the largernanoparticulate molecular target is chosen from the group consisting ofexosomes, high mw nucleic acids, including high mw DNA, oligo-nucleosomecomplexes, aggregated proteins, vesicle bound DNA, cell membranefragments and cellular debris.
 3. The method of claim 1, furthercomprising detecting a target circulating cell-free biomarker, whereinthe target circulating cell-free biomarker is chosen from the groupconsisting of mutations, deletions, rearrangements or methylated nucleicacid of circulating DNA, micro RNA, RNA from microvesicles or acombination thereof.
 4. The method of claim 3, wherein detection of thetarget circulating cell-free biomarker provides information useful forcancer diagnosis, cancer prognosis or treatment response in a patient.5. The method of claim 4, wherein the target circulating cell freebiomarker is a tumor cell-free biomarker associated with CNS tumors,neuroblastoma, gliomas, breast cancer, endometrial tumors, cervicaltumors, ovarian tumors, hepatocellular carcinoma, pancreatic carcinoma,esophageal tumors, Stoch tumors, colorectal tumors, head and necktumors, nasopharyngeal carcinoma, thyroid tumors, lymphoma, leukemia,lung cancer, non-small cell lung carcinoma, small cell lung carcinoma,testicular tumors, kidney tumors, prostate carcinoma, skin cancer,malignant melanoma, squamous cell carcinoma or a combination thereof. 6.The method of claim 5, wherein the tumor cell-free biomarker is GFAP,VEGF, EGFR, b-FGF, KRAS, YKL-40, MMP-9 or combinations thereof.
 7. Themethod of claim 1, further comprising detecting a target biomarker,wherein the target biomarker is chosen from the group consisting ofproteins, lipids, antibodies, tumor cells and nanosomes.
 8. The methodof claim 1, further comprising eluting the bound nucleic acid from thefirst chamber for further characterization.
 9. The method of claim 8,wherein the eluted nucleic acid is amplified or sequenced.
 10. Themethod of claim 1, wherein the sample is whole blood, serum, plasma,cerebrospinal fluid, body tissue, urine or saliva.
 11. The method ofclaim 1, wherein the sample is blood.
 12. The method of claim 1, whereinthe AC electrokinetic field is produced using an alternating currenthaving a voltage of 1 volt to 40 volts peak-peak, and/or a frequency of5 Hz to 5,000,000 Hz and duty cycles from 5% to 50%.
 13. The method ofclaim 1, wherein the conductivity of the fluid is greater than 500 mS/m.14. The method of claim 1, wherein the array of electrodes isspin-coated with a hydrogel having a thickness between about 0.1 micronsand 1 micron.
 15. The method of claim 5, wherein the hydrogel comprisestwo or more layers of a synthetic polymer.
 16. The method of claim 5,wherein the hydrogel has a viscosity between about 0.5 cP to about 5 cPprior to spin-coating.
 17. The method of claim 5, wherein the hydrogelhas a conductivity between about 0.1 S/m to about 1.0 S/m.
 18. Themethod of claim 1, wherein the isolated nucleic acid comprises less thanabout 10% non-nucleic acid cellular material or cellular protein bymass.
 19. The method of claim 1, wherein the array of electrodescomprises a wavy line configuration, wherein the configuration comprisesa repeating unit comprising the shape of a pair of dots connected bylinker, wherein the linker tapers inward toward the midpoint between thepair of dots, wherein the diameters of the dots are the widest pointsalong the length of the repeating unit, wherein the edge to edgedistance between a parallel set of repeating units is equidistant, orroughly equidistant.
 20. The method of claim 1, wherein the array ofelectrodes comprises a passivation layer with a relative electricalpermittivity from about 2.0 to about 4.0.
 21. A method for analyzing anucleic acid in a sample, the method comprising: a. applying a samplewith a conductivity of greater than 100 mS/m to a device, the devicecomprising an array of electrodes capable of establishing an ACelectrokinetic field region, the device further comprising at least twochambers, the sample applied to a first chamber; b. applying knownquantities of a nucleic acid standard to a second chamber; c.establishing a first AC electrokinetic high field region in the firstchamber, the first AC electrokinetic high field capable of isolatinglarger nanoparticulate molecular targets; d. establishing a second ACelectrokinetic low field in the first chamber, the second ACelectrokinetic low field capable of concentrating cells or micron-sizedentities that may be present in the sample; e. establishing a third ACelectrokinetic high field to the second chamber, the second ACelectrokinetic high field isolating the molecular nucleic acid standardapplied to the second chamber; f. flushing cells and micron-sizedentities that may be present in the sample from the first chamber; g.detecting bound nucleic acid signal on the array in the first chamberand second chamber; h. eluting the bound nucleic acid from the firstchamber; and i. performing sequencing and/or polymerase chain reactionanalysis on the eluted nucleic acid.
 22. The method of claim 21, whereinthe larger nanoparticulate molecular target is chosen from the groupconsisting of exosomes, high mw nucleic acids, including high mw DNA,oligo-nucleosome complexes, aggregated proteins, vesicle bound DNA, cellmembrane fragments and cellular debris.
 23. The method of claim 21,further comprising detecting a target circulating cell-free biomarker,wherein the target circulating cell-free biomarker is chosen from thegroup consisting of mutations, deletions, rearrangements or methylatednucleic acid of circulating DNA, micro RNA, RNA from microvesicles or acombination thereof.
 24. The method of claim 23, wherein detection ofthe target circulating cell-free biomarker provides information usefulfor cancer diagnosis, cancer prognosis or treatment response in apatient.
 25. The method of claim 24, wherein the target circulatingcell-free biomarker is a tumor cell-free biomarker associated with CNStumors, neuroblastoma, gliomas, breast cancer, endometrial tumors,cervical tumors, ovarian tumors, hepatocellular carcinoma, pancreaticcarcinoma, esophageal tumors, Stoch tumors, colorectal tumors, head andneck tumors, nasopharyngeal carcinoma, thyroid tumors, lymphoma,leukemia, lung cancer, non-small cell lung carcinoma, small cell lungcarcinoma, testicular tumors, kidney tumors, prostate carcinoma, skincancer, malignant melanoma, squamous cell carcinoma or a combinationthereof.
 26. The method of claim 25, wherein the tumor cell-freebiomarker is GFAP, VEGF, EGFR, b-FGF, KRAS, YKL-40, MMP-9 orcombinations thereof.
 27. The method of claim 21, further comprisingdetecting a target biomarker, wherein the target biomarker is chosenfrom the group consisting of proteins, lipids, antibodies, tumor cellsand nanosomes.
 28. The method of claim 2, wherein the sample is wholeblood, serum, plasma, cerebrospinal fluid, body tissue, urine or saliva.29. The method of claim 21, wherein the sample is blood.
 30. The methodof claim 21, wherein the AC electrokinetic field is produced using analternating current having a voltage of 1 volt to 40 volts peak-peak,and/or a frequency of 5 Hz to 5,000,000 Hz and duty cycles from 5% to50%.